Cationic lipids and methods for the delivery of therapeutic agents

ABSTRACT

The present invention provides compositions and methods for the delivery of therapeutic agents to cells. In particular, these include novel cationic lipids and nucleic acid-lipid particles that provide efficient encapsulation of nucleic acids and efficient delivery of the encapsulated nucleic acid in vivo. The compositions of the present invention are highly potent, thereby allowing effective know-down of a specific target protein at relatively low doses. In addition, the compositions and methods of the present invention are less toxic and provide a greater therapeutic index compared to compositions and methods previously known in the art.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/381,872, which is a 371 U.S. National Stagefiling of PCT Application No. PCT/CA2010/001029, filed Jun. 30, 2010,and claims priority to U.S. Provisional Application No. 61/222,462,filed Jul. 1, 2009, and U.S. Provisional Application No. 61/295,134,filed Jan. 14, 2010, the disclosures of which are hereby incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 27, 2014, isnamed 08155.002US2_SL.txt and is 1,436 bytes in size.

BACKGROUND OF THE INVENTION

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, andimmune-stimulating nucleic acids. These nucleic acids act via a varietyof mechanisms. In the case of interfering RNA molecules such as siRNAand miRNA, these nucleic acids can down-regulate intracellular levels ofspecific proteins through a process termed RNA interference (RNAi).Following introduction of interfering RNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the interfering RNA is displaced from the RISC complex,providing a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound interfering RNA. Havingbound the complementary mRNA, the RISC complex cleaves the mRNA andreleases the cleaved strands. RNAi can provide down-regulation ofspecific proteins by targeting specific destruction of the correspondingmRNA that encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, sinceinterfering RNA constructs can be synthesized with any nucleotidesequence directed against a target protein. To date, siRNA constructshave shown the ability to specifically down-regulate target proteins inboth in vitro and in vivo models. In addition, siRNA constructs arecurrently being evaluated in clinical studies.

However, two problems currently faced by interfering RNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as freeinterfering RNA molecules. These double-stranded constructs can bestabilized by the incorporation of chemically modified nucleotidelinkers within the molecule, e.g., phosphothioate groups. However, suchchemically modified linkers provide only limited protection fromnuclease digestion and may decrease the activity of the construct.Intracellular delivery of interfering RNA can be facilitated by the useof carrier systems such as polymers, cationic liposomes, or by thecovalent attachment of a cholesterol moiety to the molecule. However,improved delivery systems are required to increase the potency ofinterfering RNA molecules such as siRNA and miRNA and to reduce oreliminate the requirement for chemically modified nucleotide linkers.

In addition, problems remain with the limited ability of therapeuticnucleic acids such as interfering RNA to cross cellular membranes (see,Vlassov et al., Biochim. Biophys. Acta, 1197:95-1082 (1994)) and in theproblems associated with systemic toxicity, such as complement-mediatedanaphylaxis, altered coagulatory properties, and cytopenia (Galbraith etal., Antisense Nucl. Acid Drug Des., 4:201-206 (1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. Zelphati et al. (J. Contr. Rel., 41:99-119(1996)) describes the use of anionic (conventional) liposomes, pHsensitive liposomes, immunoliposomes, fusogenic liposomes, and cationiclipid/antisense aggregates. Similarly, siRNA has been administeredsystemically in cationic liposomes, and these nucleic acid-lipidparticles have been reported to provide improved down-regulation oftarget proteins in mammals including non-human primates (Zimmermann etal., Nature, 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these nucleic acid-lipid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel cationic (amino) lipids and lipidparticles comprising these lipids, which are advantageous for the invivo delivery of nucleic acids, as well as nucleic acid-lipid particlecompositions suitable for in vivo therapeutic use. The present inventionalso provides methods of making these compositions, as well as methodsof introducing nucleic acids into cells using these compositions, e.g.,for the treatment of various disease conditions.

In one aspect, the present invention provides a cationic lipid ofFormula I having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either absent or present and when present are        either the same or different and are independently an optionally        substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl; and    -   n is 0, 1, 2, 3, or 4.

In a particular embodiment, the cationic lipid of Formula I has thestructure:

In another aspect, the present invention provides a cationic lipid ofFormula II having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least three sites of unsaturation or a        substituted C₁₂-C₂₄ alkyl;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and

Y and Z are either the same or different and are independently O, S, orNH.

In particular embodiments, the cationic lipid of Formula II has astructure selected from the group consisting of

In yet another aspect, the present invention provides a cationic lipidof Formula III having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are joined to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula III has astructure selected from the group consisting of:

In still yet another aspect, the present invention provides a cationiclipid of Formula IV having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula IV has astructure selected from the group consisting of

In another aspect, the present invention provides a cationic lipid ofFormula V having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are different and are independently an optionally        substituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, or        C₁-C₂₄ acyl; and

-   n is 0, 1, 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula V is anasymmetric lipid having a structure selected from the group consistingof:

In yet another aspect, the present invention provides a cationic lipidof Formula VI having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least four sites of unsaturation or a        substituted C₁₂-C₂₄ alkyl; and    -   n is 0, 1, 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula VI has astructure selected from the group consisting of

In still yet another aspect, the present invention provides a cationiclipid of Formula VII having the following structure:

or salts thereof, wherein:

-   -   R¹ is hydrogen (H) or —(CH₂)_(q)—NR⁶R⁷R⁸, wherein:        -   R⁶ and R⁷ are either the same or different and are            independently an optionally substituted C₁-C₆ alkyl, C₂-C₆            alkenyl, or C₂-C₆ alkynyl, or R⁶ and R⁷ may join to form an            optionally substituted heterocyclic ring of 4 to 6 carbon            atoms and 1 or 2 heteroatoms selected from the group            consisting of nitrogen (N), oxygen (O), and mixtures            thereof;        -   R⁸ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to            provide a quaternary amine; and        -   q is 0, 1, 2, 3, or 4;    -   R² is an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or        C₂-C₆ alkynyl;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula VII has astructure selected from the group consisting of:

In another aspect, the present invention provides a cationic lipid ofFormula VIII having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴, R⁵, and R⁶ are either the same or different and are        independently an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄        alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula VIII has astructure selected from the group consisting of:

In yet another aspect, the present invention provides a cationic lipidof Formula IX having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH,    -   with the proviso that if q is 1, R¹ and R² are not both methyl        groups when R⁴ and R⁵ are both linoleyl moieties, Y and Z are        both 0, and the alkylamino group is attached to the ‘5’ position        of the 6-membered ring.

In a particular embodiment, the cationic lipid of Formula IX has thestructure:

In still yet another aspect, the present invention provides a cationiclipid of Formula X having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least one site of unsaturation in the trans (E)        configuration;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH.

In a particular embodiment, the cationic lipid of Formula X has thestructure:

In another aspect, the present invention provides a cationic lipid ofFormula XI having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are joined to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH.

In a particular embodiment, the cationic lipid of Formula XI has thestructure:

In yet another aspect, the present invention provides a cationic lipidof Formula XII having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;

R⁴ and R⁵ are either the same or different and are independently asubstituted C₁₂-C₂₄ alkyl; and

-   -   n is 0, 1, 2, 3, or 4.

In a particular embodiment, the cationic lipid of Formula XII has astructure selected from the group consisting of:

In a further aspect, the present invention provides a lipid particlecomprising one or more of the above cationic lipids of Formula I-XII orsalts thereof. In certain embodiments, the lipid particle furthercomprises one or more non-cationic lipids such as neutral lipids. Incertain other embodiments, the lipid particle further comprises one ormore conjugated lipids capable of reducing or inhibiting particleaggregation. In additional embodiments, the lipid particle furthercomprises one or more active agents or therapeutic agents.

In certain embodiments, the non-cationic lipid component of the lipidparticle may comprise a phospholipid, cholesterol (or cholesterolderivative), or a mixture thereof. In one particular embodiment, thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof. In certainother embodiments, the conjugated lipid component of the lipid particlemay comprise a polyethyleneglycol (PEG)-lipid conjugate. In oneparticular embodiment, the PEG-lipid conjugate comprises aPEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA)conjugate, or a mixture thereof.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA,Dicer-substrate dsRNA, shRNA, or mixtures thereof. In certain otherinstances, the nucleic acid comprises single-stranded or double-strandedDNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisenseoligonucleotide, a ribozyme, a plasmid, an immunostimulatoryoligonucleotide, or mixtures thereof.

In other embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particle such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In further embodiments, the lipid particle is substantiallynon-toxic to mammals such as humans.

In preferred embodiments, the present invention provides serum-stablenucleic acid-lipid particles (SNALP) comprising: (a) one or more nucleicacids such as interfering RNA molecules; (b) one or more cationic lipidsof Formula I-XII or salts thereof; (c) one or more non-cationic lipids;and (d) one or more conjugated lipids that inhibit aggregation ofparticles.

In some embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more nucleic acids; (b)one or more cationic lipids of Formula I-XII or salts thereof comprisingfrom about 50 mol % to about 85 mol % of the total lipid present in theparticle; (c) one or more non-cationic lipids comprising from about 13mol % to about 49.5 mol % of the total lipid present in the particle;and (d) one or more conjugated lipids that inhibit aggregation ofparticles comprising from about 0.5 mol % to about 2 mol % of the totallipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I-XII ora salt thereof comprising from about 52 mol % to about 62 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 36 mol % toabout 47 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 2 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “1:57”formulation. In one particular embodiment, the 1:57 formulation is afour-component system comprising about 1.4 mol % PEG-lipid conjugate(e.g., PEG2000-C-DMA), about 57.1 mol % cationic lipid of Formula I-XIIor a salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol %cholesterol (or derivative thereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I-XII ora salt thereof comprising from about 56.5 mol % to about 66.5 mol % ofthe total lipid present in the particle; (c) cholesterol or a derivativethereof comprising from about 31.5 mol % to about 42.5 mol % of thetotal lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 1 mol % to about 2 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “1:62” formulation. In oneparticular embodiment, the 1:62 formulation is a three-component systemwhich is phospholipid-free and comprises about 1.5 mol % PEG-lipidconjugate (e.g., PEG2000-C-DMA), about 61.5 mol % cationic lipid ofFormula I-XII or a salt thereof, and about 36.9 mol % cholesterol (orderivative thereof).

Additional embodiments related to the 1:57 and 1:62 formulations aredescribed in PCT Publication No. WO 09/127,060 and U.S. application Ser.No. 12/794,701, filed Jun. 4, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

In other embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more nucleic acids; (b)one or more cationic lipids of Formula I-XII or salts thereof comprisingfrom about 2 mol % to about 50 mol % of the total lipid present in theparticle; (c) one or more non-cationic lipids comprising from about 5mol % to about 90 mol % of the total lipid present in the particle; and(d) one or more conjugated lipids that inhibit aggregation of particlescomprising from about 0.5 mol % to about 20 mol % of the total lipidpresent in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I-XII ora salt thereof comprising from about 30 mol % to about 50 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 47 mol % toabout 69 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 3 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “2:40”formulation. In one particular embodiment, the 2:40 formulation is afour-component system which comprises about 2 mol % PEG-lipid conjugate(e.g., PEG2000-C-DMA), about 40 mol % cationic lipid of Formula I-XII ora salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol %cholesterol (or derivative thereof).

In further embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) comprising: (a) one or more nucleicacids; (b) one or more cationic lipids of Formula I-XII or salts thereofcomprising from about 50 mol % to about 65 mol % of the total lipidpresent in the particle; (c) one or more non-cationic lipids comprisingfrom about 25 mol % to about 45 mol % of the total lipid present in theparticle; and (d) one or more conjugated lipids that inhibit aggregationof particles comprising from about 5 mol % to about 10 mol % of thetotal lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I-XII ora salt thereof comprising from about 50 mol % to about 60 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 35 mol % toabout 45 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 5 mol % to about 10 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “7:54”formulation. In certain instances, the non-cationic lipid mixture in the7:54 formulation comprises: (i) a phospholipid of from about 5 mol % toabout 10 mol % of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 35mol % of the total lipid present in the particle. In one particularembodiment, the 7:54 formulation is a four-component system whichcomprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about54 mol % cationic lipid of Formula I-XII or a salt thereof, about 7 mol% DPPC (or DSPC), and about 32 mol % cholesterol (or derivativethereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I-XII ora salt thereof comprising from about 55 mol % to about 65 mol % of thetotal lipid present in the particle; (c) cholesterol or a derivativethereof comprising from about 30 mol % to about 40 mol % of the totallipid present in the particle; and (d) a PEG-lipid conjugate comprisingfrom about 5 mol % to about 10 mol % of the total lipid present in theparticle. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “7:58” formulation. In one particularembodiment, the 7:58 formulation is a three-component system which isphospholipid-free and comprises about 7 mol % PEG-lipid conjugate (e.g.,PEG750-C-DMA), about 58 mol % cationic lipid of Formula I-XII or a saltthereof, and about 35 mol % cholesterol (or derivative thereof).

Additional embodiments related to the 7:54 and 7:58 formulations aredescribed in U.S. application Ser. No. 12/828,189, entitled “Novel LipidFormulations for Delivery of Therapeutic Agents to Solid Tumors,” filedJun. 30, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The present invention also provides pharmaceutical compositionscomprising a lipid particle such as a nucleic acid-lipid particle (e.g.,SNALP) and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods forintroducing one or more therapeutic agents such as nucleic acids into acell, the method comprising contacting the cell with a lipid particledescribed herein (e.g., SNALP). In one embodiment, the cell is in amammal and the mammal is a human.

In yet another aspect, the present invention provides methods for the invivo delivery of one or more therapeutic agents such as nucleic acids,the method comprising administering to a mammal a lipid particledescribed herein (e.g., SNALP). In certain embodiments, the lipidparticles (e.g., SNALP) are administered by one of the following routesof administration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intraarticular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the lipidparticles (e.g., SNALP) are administered systemically, e.g., via enteralor parenteral routes of administration. In preferred embodiments, themammal is a human.

In a further aspect, the present invention provides methods for treatinga disease or disorder in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of alipid particle (e.g., SNALP) comprising one or more therapeutic agentssuch as nucleic acids. Non-limiting examples of diseases or disordersinclude a viral infection, a liver disease or disorder, and cancer.Preferably, the mammal is a human.

In certain embodiments, the present invention provides methods fortreating a liver disease or disorder by administering a nucleic acidsuch as an interfering RNA (e.g., siRNA) in nucleic acid-lipid particles(e.g., SNALP), alone or in combination with a lipid-lowering agent.Examples of lipid diseases and disorders include, but are not limitedto, dyslipidemia (e.g., hyperlipidemias such as elevated triglyceridelevels (hypertriglyceridemia) and/or elevated cholesterol levels(hypercholesterolemia)), atherosclerosis, coronary heart disease,coronary artery disease, atherosclerotic cardiovascular disease (CVD),fatty liver disease (hepatic steatosis), abnormal lipid metabolism,abnormal cholesterol metabolism, diabetes (including Type 2 diabetes),obesity, cardiovascular disease, and other disorders relating toabnormal metabolism. Non-limiting examples of lipid-lowering agentsinclude statins, fibrates, ezetimibe, thiazolidinediones, niacin,beta-blockers, nitroglycerin, calcium antagonists, and fish oil.

In one particular embodiment, the present invention provides a methodfor lowering or reducing cholesterol levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood cholesterol levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) described herein comprising one or more interfering RNAs(e.g., siRNAs) that target one or more genes associated with metabolicdiseases and disorders. In another particular embodiment, the presentinvention provides a method for lowering or reducing triglyceride levelsin a mammal (e.g., human) in need thereof (e.g., a mammal with elevatedblood triglyceride levels), the method comprising administering to themammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., a SNALP formulation) described herein comprising one ormore interfering RNAs (e.g., siRNAs) that target one or more genesassociated with metabolic diseases and disorders. These methods can becarried out in vitro using standard tissue culture techniques or in vivoby administering the interfering RNA (e.g., siRNA) using any means knownin the art. In preferred embodiments, the interfering RNA (e.g., siRNA)is delivered to a liver cell (e.g., hepatocyte) in a mammal such as ahuman.

Additional embodiments related to treating a liver disease or disorderusing a lipid particle are described in, e.g., PCT Application No.PCT/CA2010/000120, filed Jan. 26, 2010, and U.S. Patent Publication No.20060134189, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

In other embodiments, the present invention provides methods fortreating a cell proliferative disorder such as cancer by administering anucleic acid such as an interfering RNA (e.g., siRNA) in nucleicacid-lipid particles (e.g., SNALP), alone or in combination with achemotherapy drug. The methods can be carried out in vitro usingstandard tissue culture techniques or in vivo by administering theinterfering RNA (e.g., siRNA) using any means known in the art. Inpreferred embodiments, the interfering RNA (e.g., siRNA) is delivered toa cancer cell in a mammal such as a human, alone or in combination witha chemotherapy drug. The nucleic acid-lipid particles and/orchemotherapy drugs may also be co-administered with conventionalhormonal, immunotherapeutic, and/or radiotherapeutic agents.

Additional embodiments related to treating a cell proliferative disorderusing a lipid particle are described in, e.g., PCT Publication No. WO09/082,817, U.S. Patent Publication No. 20090149403, PCT Publication No.WO 09/129,319, and U.S. Provisional Application No. 61/245,143, filedSep. 23, 2009, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

In further embodiments, the present invention provides methods forpreventing or treating a viral infection such as an Ebola virusinfection by administering a nucleic acid such as an interfering RNA(e.g., siRNA) in nucleic acid-lipid particles (e.g., SNALP), alone or incombination with the administration of conventional agents used to treator ameliorate the viral condition or any of the symptoms associatedtherewith. The methods can be carried out in vitro using standard tissueculture techniques or in vivo by administering the interfering RNA usingany means known in the art. In preferred embodiments, the interferingRNA (e.g., siRNA) is delivered to cells, tissues, or organs of a mammalsuch as a human that are infected and/or susceptible of being infectedwith the virus.

Additional embodiments related to preventing or treating a viralinfection using a lipid particle are described in, e.g., U.S. PatentPublication No. 20070218122, U.S. Patent Publication No. 20070135370,U.S. Provisional Application No. 61/286,741, filed Dec. 15, 2009, PCTApplication No. PCT/CA2010/000444, entitled “Compositions and Methodsfor Silencing Hepatitis C Virus Expression,” filed Mar. 19, 2010, andU.S. Provisional Application No. 61/319,855, filed Mar. 31, 2010, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

The lipid particles of the invention (e.g., SNALP) comprising one ormore cationic lipids of Formula I-XII or salts thereof are particularlyadvantageous and suitable for use in the administration of nucleic acidssuch as interfering RNA to a subject (e.g., a mammal such as a human)because they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites, andare capable of reaching target cell populations.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the apparent pK_(a) values of exemplary SNALP formulationscontaining various cationic lipids described herein.

FIG. 2 shows the apparent pK_(a) values of additional exemplary SNALPformulations containing various cationic lipids described herein.

FIG. 3 shows a comparison of the plasma total cholesterol knockdownefficacy of exemplary SNALP formulations containing various cationiclipids described herein.

FIG. 4 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary SNALP formulations containing various cationic lipidsdescribed herein.

FIG. 5 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary SNALP formulations containing various cationiclipids described herein.

FIG. 6 shows a dose response evaluation of three different doses ofexemplary SNALP formulations containing various cationic lipidsdescribed herein on liver ApoB mRNA knockdown activity.

FIG. 7 shows the tolerability of an exemplary γ-DLenDMA SNALPformulation.

FIG. 8 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary SNALP formulations containing various cationiclipids described herein.

FIG. 9 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary SNALP formulations containing various cationiclipids described herein.

FIG. 10 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary SNALP formulations containing various cationiclipids described herein.

FIG. 11 shows a comparison of the tumor PLK-1 mRNA knockdown activity ofexemplary SNALP formulations containing various cationic lipidsdescribed herein.

FIG. 12 shows the effect of ApoE−/− or C57 serum pre-incubation onsilencing activity with exemplary SNALP formulations containing variouscationic lipids described herein.

FIG. 13 shows the effect of ApoE−/− or C57 serum pre-incubation onsilencing activity with additional exemplary SNALP formulationscontaining various cationic lipids described herein.

FIG. 14 shows the effect of ApoE−/− or C57 serum pre-incubation onsilencing activity with additional exemplary SNALP formulationscontaining various cationic lipids described herein.

FIG. 15 shows the in vitro serum protein binding to SNALP followingpre-incubation with C57BL/6, APOE−/−, or LDLr−/− serum. (a) SNALPcontaining 0.1 mol % Biotin-PEG-DSPE were pre-incubated with mouse serum(C57BL/6 or APOE−/−) for 1 hour at 37° C. Proteins were then extracted.For each serum type, 2 discrete SNALP preparations were used (‘A’ and‘B’). Lane 1: SNALP ‘A’+C57BL/06 serum. Lane 2: SNALP ‘B’+C57BL/6 serum.Lane 3: C57BL/6 serum control. Lane 4: SNALP ‘A’+APOE−/− serum. Lane 5:SNALP ‘B’+APOE−/− serum. Lane 6: APOE−/− serum control. Major proteinswere identified by LC/MS and Western blot analysis. (b) SNALP ligandblotting. SNALP containing 0.1 mol % Biotin-PEG-DSPE at equivalent lipidconcentration were pre-incubated with APOE containing C57BL/6 serum(Blots 1, 4), APOE−/− serum (Blots 2, 5), or in the absence of serum(Blots 3, 6) for 1 hour at 37° C. Binding to liver proteins on theC57BL/6 (Blots 1 to 3) and LDLr−/− (Blots 4 to 6) was determined usingstreptavidin-HRP. APOE, apolipoprotein E; LDLr, low-density-lipoproteinreceptor; SNALP, stable nucleic acid lipid particles.

FIG. 16 shows the in vitro uptake and associated gene-silencing withSNALP by primary hepatocytes isolated from C57BL/6, APOE−/−, and LDLr−/−mice. (a, b) ³H-CHE labeled SNALP were pre-incubated with mouse serum(C57BL/6, APOE−/− or LDLr−/−) for 1 hour at 37° C. Hepatocytes wereexposed to SNALP for either 4 hours (a) or 24 hours (b) at aconcentration of 0.25 μg/mL siRNA. SNALP uptake was normalized to thetotal protein content. Error bars represent SD, n=3. (c, d) UnlabeledSNALP, pre-incubated in serum, were used to treat hepatocytes.Hepatocytes were exposed to SNALP for either 4 hours (c) or 24 hours (d)at a concentration of 0.25 μg/mL siRNA. The APOB and GAPDH mRNAconcentrations were determined. Results expressed as % relative to aSNALP mismatch control. Error bars represent SD, n=3. APOE,apolipoprotein E; LDLr, low-density-lipoprotein receptor; SNALP, stablenucleic acid lipid particles.

FIG. 17 shows the in vivo clearance, tissue biodistribution, andassociated ApoB gene-silencing of SNALP in C57BL/6, APOE−/−, and LDLr−/−mice. (a) Clearance of ³H-CHE labeled SNALP in C57BL/6, APOE−/−, andLDLr−/− mice. Whole blood was obtained from tail nicks, and ³H countswere used to determine the percentage of injected dose remaining. (b)Biodistribution of ³H-CHE labeled SNALP in C57BL/6, APOE−/−, and LDLr−/−mice. Accumulation was determined 24 hours after injection. (c) In vivoknockdown of APOB mRNA. Liver ApoB and GAPDH mRNA were measured 48 hoursfollowing SNALP treatment. The ApoB:GAPDH ratio was determined andreported relative to PBS controls. All mice received a singleintravenous administration of SNALP via the lateral tail vein. Errorbars represent SD, n=3 for each experiment. APOB, apolipoprotein B;APOE, apolipoprotein E; LDLr, GAPDH, glyceraldehyde 3-phosphatedehydrogenase; low-density-lipoprotein receptor; PBS, phosphate-bufferedsaline; SNALP, stable nucleic acid lipid particles.

FIG. 18 shows the effect of human serum components on in vitro APOB mRNAknockdown in primary hepatocytes. (a) Effect of human lipoprotein/SNALPpre-incubation on knockdown in APOE−/− mouse hepatocytes. SNALP werepre-incubated with human lipoproteins or mouse or human sera for 1 hourat 37° C. APOE−/− hepatocytes were then exposed to SNALP for 24 hours.(b) Gene-silencing in human primary hepatocytes. Human hepatocytes wereexposed to SNALP (containing antiAPOB or the nontargeting antiLuc siRNA)pre-incubated with or without human serum at a siRNA concentration of0.125 μg/mL. (c) Effect of APOE isoform on SNALP-mediated gene silencingin human hepatocytes. SNALP were pre-incubated with each of the 3isoforms of human APOE. Human hepatocytes were then exposed to the SNALPfor varying durations at a siRNA concentration of 0.125 μg/mL. The APOBand GAPDH mRNA concentrations were determined and expressed as a ratiorelative to PBS control. Error bars represent SD, n=3. APOE,apolipoprotein E; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA,messenger ribonucleic acid; PBS, phosphate-buffered saline; siRNA, smallinterfering ribonucleic acid; SNALP, stable nucleic acid lipidparticles.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular, thepresent invention provides nucleic acid-lipid particle compositionscomprising one or more of the novel cationic lipids described hereinthat provide increased activity of the nucleic acid (e.g., interferingRNA) and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid particle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of interfering RNA such as siRNA. It is shownherein that these improved lipid particle compositions are effective indown-regulating (e.g., silencing) the protein levels and/or mRNA levelsof target genes. Furthermore, it is shown herein that the activity ofthese improved lipid particle compositions is dependent on the presenceof the novel cationic lipids of the invention.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, the present inventionfurther provides methods of treating diseases or disorders in a subjectin need thereof by contacting the subject with a lipid particle thatencapsulates or is associated with a suitable therapeutic agent, whereinthe lipid particle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,interfering RNA molecules such as siRNA. Therefore, the lipid particlesand compositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle comprising one or more novelcationic lipids described herein, wherein the lipid particleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an siRNA). Alternatively, the lipid particles andcompositions of the present invention may be used to increase theexpression of a desired protein both in vitro and in vivo by contactingcells with a lipid particle comprising one or more novel cationic lipidsdescribed herein, wherein the lipid particle encapsulates or isassociated with a nucleic acid that enhances expression of the desiredprotein (e.g., a plasmid encoding the desired protein).

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid particles and compositions comprising the same, andtheir use to deliver active or therapeutic agents such as nucleic acidsto modulate gene and protein expression, are described in further detailbelow.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO2004/104199) that is capable of reducing or inhibiting the expression ofa target gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule. As used herein, theterm “siRNA” includes RNA-RNA duplexes as well as DNA-RNA hybrids (see,e.g., PCT Publication No. WO 2004/078941).

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of an interfering RNA (e.g., siRNA) to silence, reduce, orinhibit the expression of a target gene. To examine the extent of genesilencing, a test sample (e.g., a sample of cells in culture expressingthe target gene) or a test mammal (e.g., a mammal such as a human or ananimal model such as a rodent (e.g., mouse) or a non-human primate(e.g., monkey) model) is contacted with an interfering RNA (e.g., siRNA)that silences, reduces, or inhibits expression of the target gene.Expression of the target gene in the test sample or test animal iscompared to expression of the target gene in a control sample (e.g., asample of cells in culture expressing the target gene) or a controlmammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or non-human primate (e.g., monkey) model) that isnot contacted with or administered the interfering RNA (e.g., siRNA).The expression of the target gene in a control sample or a controlmammal may be assigned a value of 100%. In particular embodiments,silencing, inhibition, or reduction of expression of a target gene isachieved when the level of target gene expression in the test sample orthe test mammal relative to the level of target gene expression in thecontrol sample or the control mammal is about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or0%. In other words, the interfering RNA (e.g., siRNA) silences, reduces,or inhibits the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the interfering RNA (e.g., siRNA).Suitable assays for determining the level of target gene expressioninclude, without limitation, examination of protein or mRNA levels usingtechniques known to those of skill in the art, such as, e.g., dot blots,Northern blots, in situ hybridization, ELISA, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as an interfering RNA is an amountsufficient to produce the desired effect, e.g., an inhibition ofexpression of a target sequence in comparison to the normal expressionlevel detected in the absence of an interfering RNA. Inhibition ofexpression of a target gene or target sequence is achieved when thevalue obtained with an interfering RNA relative to the control is about95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expressionof a target gene or target sequence include, e.g., examination ofprotein or RNA levels using techniques known to those of skill in theart such as dot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, as well as phenotypic assays knownto those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, IL-8, or IL-12) by a respondercell in vitro or a decrease in cytokine production in the sera of amammalian subject after administration of the interfering RNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-γ,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, TGF, andcombinations thereof. Detectable immune responses also include, e.g.,induction of interferon-induced protein with tetratricopeptide repeats 1(IFIT1) mRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

Non-limiting examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Anotherexample is a global alignment algorithm for determining percent sequenceidentity such as the Needleman-Wunsch algorithm for aligning protein ornucleotide (e.g., RNA) sequences.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNAduplexes, pre-condensed DNA, a PCR product, vectors (P1, PAC, BAC, YAC,artificial chromosomes), expression cassettes, chimeric sequences,chromosomal DNA, or derivatives and combinations of these groups. RNAmay be in the form of small interfering RNA (siRNA), Dicer-substratedsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA),microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), andcombinations thereof. Nucleic acids include nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,and which have similar binding properties as the reference nucleic acid.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), to a target site of interest (e.g., cell,tissue, organ, and the like). In preferred embodiments, the lipidparticle of the invention is a nucleic acid-lipid particle, which istypically formed from a cationic lipid, a non-cationic lipid, andoptionally a conjugated lipid that prevents aggregation of the particle.In other preferred embodiments, the active agent or therapeutic agent,such as a nucleic acid, may be encapsulated in the lipid portion of theparticle, thereby protecting it from enzymatic degradation.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle), wherein the nucleic acid(e.g., an interfering RNA) is fully encapsulated within the lipid. Incertain instances, SNALP are extremely useful for systemic applications,as they can exhibit extended circulation lifetimes following intravenous(i.v.) injection, they can accumulate at distal sites (e.g., sitesphysically separated from the administration site), and they can mediatesilencing of target gene expression at these distal sites. The nucleicacid may be complexed with a condensing agent and encapsulated within aSNALP as set forth in PCT Publication No. WO 00/03683, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The lipid particles of the invention (e.g., SNALP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Patent Publication Nos. 20040142025 and 20070042031, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), with full encapsulation, partialencapsulation, or both. In a preferred embodiment, the nucleic acid isfully encapsulated in the lipid particle (e.g., to form a SNALP or othernucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,and U.S. Provisional Application No. 61/295, 140, filed Jan. 14, 2010),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused. The disclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulthydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa SNALP, to fuse with the membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as SNALPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site such as a tumor or other targetsite such as a site of inflammation or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,liver cancer, lung cancer, colon cancer, rectal cancer, anal cancer,bile duct cancer, small intestine cancer, stomach (gastric) cancer,esophageal cancer; gallbladder cancer, pancreatic cancer, appendixcancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer,renal cancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein., a “tumor” comprises one or more cancerous cells.

III. Novel Cationic Lipids

The present invention provides, inter alia, novel cationic (amino)lipids that can advantageously be used in the lipid particles describedherein for the in vitro and/or in vivo delivery of therapeutic agentssuch as nucleic acids to cells. The novel cationic lipids of theinvention have the structures set forth in Formulas I-XII herein, andinclude the (R) and/or (S) enantiomers thereof.

In some embodiments, the cationic lipid comprises a racemic mixture. Inother embodiments, the cationic lipid comprises a mixture of one or morediastereomers. In certain embodiments, the cationic lipid is enriched inone enantiomer, such that the cationic lipid comprises at least about55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. Incertain other embodiments, the cationic lipid is enriched in onediastereomer, such that the cationic lipid comprises at least about 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. Incertain additional embodiments, the cationic lipid is chirally pure(e.g., comprises a single optical isomer). In further embodiments, thecationic lipid is enriched in one optical isomer (e.g., an opticallyactive isomer), such that the cationic lipid comprises at least about55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomeric excess. Thepresent invention provides the synthesis of the cationic lipids ofFormulas I-XII as a racemic mixture or in optically pure form.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids.

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid disclosed herein and one or moreanions. Non-limiting examples of anions include inorganic and organicanions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate(e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate,dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite,nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate,hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate,lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, whileunsaturated cyclic alkyls include, without limitation, cyclopentenyl,cyclohexenyl, and the like.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” mean that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O), two hydrogen atoms are replaced.In this regard, substituents include, but are not limited to, oxo,halogen, heterocycle, —CN, —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, R^(x) andR^(y) are the same or different and are independently hydrogen, alkyl,or heterocycle, and each of the alkyl and heterocycle substituents maybe further substituted with one or more of oxo, halogen, —OH, —CN,alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)OR^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term “optionally substituted,”when used before a list of substituents, means that each of thesubstituents in the list may be optionally substituted as describedherein.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

In one aspect, the present invention provides a cationic lipid ofFormula I having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either absent or present and when present are        either the same or different and are independently an optionally        substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl; and n is 0, 1, 2, 3,        or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, R⁴ and R⁵ are both butyl groups. In yet another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pH of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substituted C₂-C₆or C₂-C₄ alkyl or C₂-C₆ or C₂-C₄ alkenyl.

In an alternative embodiment, the cationic lipid of Formula I comprisesester linkages between the amino head group and one or both of the alkylchains. In some embodiments, the cationic lipid of Formula I forms asalt (preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

Although each of the alkyl chains in Formula I contains cis double bondsat positions 6, 9, and 12 (i.e., cis,cis,cis-Δ⁶,Δ⁹,Δ¹²), in analternative embodiment, one, two, or three of these double bonds in oneor both alkyl chains may be in the trans configuration.

In a particularly preferred embodiment, the cationic lipid of Formula Ihas the structure:

In another aspect, the present invention provides a cationic lipid ofFormula II having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least three sites of unsaturation or a        substituted C₁₂-C₂₄ alkyl;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C4 alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.In other preferred embodiments, R⁴ and R⁵ are both phytanyl moieties.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety.

In embodiments where at least one of R⁴ and R⁵ comprises at least threesites of unsaturation, the double bonds present in one or both alkylchains may be in the cis and/or trans configuration. In someembodiments, R⁴ and R⁵ are independently selected from the groupconsisting of a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, and a phytanyl moiety, as well as acyl derivatives thereof(e.g., linolenoyl, γ-linolenoyl, phytanoyl, etc.). In certain instances,the octadecatrienyl moiety is a linolenyl moiety or a γ-linolenylmoiety. In preferred embodiments, R⁴ and R⁵ are both linolenyl moietiesor γ-linolenyl moieties. In particular embodiments, R⁴ and R⁵independently comprise a backbone of from about 16 to about 22 carbonatoms, and one or both of R⁴ and R⁵ independently comprise at leastthree, four, five, or six sites of =saturation.

In some embodiments, the cationic lipid of Formula II forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula II is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula IIhas a structure selected from the group consisting of:

In yet another aspect, the present invention provides a cationic lipidof Formula III having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are joined to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are joined to form a heterocyclic ring of5 carbon atoms and 1 nitrogen atom. In certain instances, theheterocyclic ring is substituted with a substituent such as a hydroxylgroup at the ortho, meta, and/or para positions. In a preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a), of the cationic lipid and R³ is hydrogen when the pHis below the pK_(a) of the cationic lipid such that the amino head groupis protonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,phytanoyl, etc.). In some instances, the octadecadienyl moiety is alinoleyl moiety. In other instances, the octadecatrienyl moiety is alinolenyl moiety or a γ-linolenyl moiety. In particular embodiments, R⁴and R⁵ are both linoleyl moieties, linolenyl moieties, γ-linolenylmoieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula III forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula III is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaIII has a structure selected from the group consisting of:

In still yet another aspect, the present invention provides a cationiclipid of Formula IV having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula IV forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula IV is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula IVhas a structure selected from the group consisting of:

In another aspect, the present invention provides a cationic lipid ofFormula V having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are different and are independently an optionally        substituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, or        C₁-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are different and are independently an optionallysubstituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl, C₄-C₂₀ alkynyl, or C₄-C₂₀acyl.

In some embodiments, R⁴ is an optionally substituted C₁₂-C₂₄ alkyl,C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, and R⁵ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, orC₄-C₁₀ acyl. In certain instances, R⁴ is an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is an optionally substitutedC₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ or C₆ alkynyl, or C₄-C₈ orC₆ acyl.

In other embodiments, R⁴ is an optionally substituted C₄-C₁₀ alkyl,C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl. In certain instances, R⁴ is an optionally substituted C₄-C₈ or C₆alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl,and R⁵ is an optionally substituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ orC₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In particular embodiments, R⁴ is a linoleyl moiety, and R⁵ is a C₆ alkylmoiety, a C₆ alkenyl moiety, an octadecyl moiety, an oleyl moiety, alinolenyl moiety, a γ-linolenyl moiety, or a phytanyl moiety. In otherembodiments, one of R⁴ or R⁵ is a phytanyl moiety.

In some embodiments, the cationic lipid of Formula V forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula V is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula Vis an asymmetric lipid having a structure selected from the groupconsisting of:

In yet another aspect, the present invention provides a cationic lipidof Formula VI having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least four sites of unsaturation or a        substituted C₁₂-C₂₄ alkyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety.

In embodiments where at least one of R⁴ and R⁵ comprises at least foursites of unsaturation, the double bonds present in one or both alkylchains may be in the cis and/or trans configuration. In a particularembodiment, R⁴ and R⁵ independently comprise four, five, or six sites ofunsaturation. In some instances, R⁴ comprises four, five, or six sitesof unsaturation and R⁵ comprises zero, one, two, three, four, five, orsix sites of unsaturation. In other instances, R⁴ comprises zero, one,two, three, four, five, or six sites of unsaturation and R⁵ comprisesfour, five, or six sites of unsaturation. In a preferred embodiment,both R⁴ and R⁵ comprise four, five, or six sites of unsaturation. Inparticular embodiments, R⁴ and R⁵ independently comprise a backbone offrom about 18 to about 24 carbon atoms, and one or both of R⁴ and R⁵independently comprise at least four, five, or six sites ofunsaturation.

In some embodiments, the cationic lipid of Formula VI forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VI is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula VIhas a structure selected from the group consisting of:

In still yet another aspect, the present invention provides a cationiclipid of Formula VII having the following structure:

or salts thereof, wherein:

-   -   R¹ is hydrogen (H) or —(CH₂)_(q)—NR⁶R⁷R⁸, wherein:        -   R⁶ and R⁷ are either the same or different and are            independently an optionally substituted C₁-C₆ alkyl, C₂-C₆            alkenyl, or C₂-C₆ alkynyl, or R⁶ and R⁷ may join to form an            optionally substituted heterocyclic ring of 4 to 6 carbon            atoms and 1 or 2 heteroatoms selected from the group            consisting of nitrogen (N), oxygen (O), and mixtures            thereof;        -   R⁸ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to            provide a quaternary amine; and        -   q is 0, 1, 2, 3, or 4;    -   R² is an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or        C₂-C₆ alkynyl;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R² is an optionally substituted C₁-C₄ alkyl, C₂-C₄alkenyl, or C₂-C₄ alkynyl. In other embodiments, R³ is absent when thepH is above the pK_(a) of the cationic lipid and R³ is hydrogen when thepH is below the pK_(a) of the cationic lipid such that the amino headgroup is protonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In certainembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In further embodiments, R⁶ and R⁷ are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In otherembodiments, R⁸ is absent when the pH is above the pK_(a) of thecationic lipid and R⁸ is hydrogen when the pH is below the pK_(a) of thecationic lipid such that the amino head group is protonated. In analternative embodiment, R⁸ is an optionally substituted C₁-C₄ alkyl toprovide a quaternary amine.

In a preferred embodiment, R¹ is hydrogen and R² is an ethyl group. Inanother preferred embodiment, R⁶ and R⁷ are both methyl groups. Incertain instances, n is 1. In certain other instances, q is 1.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula VII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaVII has a structure selected from the group consisting of:

In another aspect, the present invention provides a cationic lipid ofFormula VIII having the following structure:

or salts thereof, wherein:

R¹ and R² are either the same or different and are independently anoptionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, orR¹ and R² may join to form an optionally substituted heterocyclic ringof 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the groupconsisting of nitrogen (N), oxygen (O), and mixtures thereof;

-   -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴, R⁵, and R⁶ are either the same or different and are        independently an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄        alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In one particularembodiment, n is 1. In another particular embodiment, n is 2. In yetanother particular embodiment, n is 3. In other embodiments, R³ isabsent when the pH is above the pK_(a) of the cationic lipid and R³ ishydrogen when the pH is below the pK_(a) of the cationic lipid such thatthe amino head group is protonated. In an alternative embodiment, R³ isan optionally substituted C₁-C₄ alkyl to provide a quaternary amine. Infurther embodiments, R⁴, R⁵, and R⁶ are independently an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴, R⁵, and R⁶ are independently selected fromthe group consisting of a dodecadienyl moiety, a tetradecadienyl moiety,a hexadecadienyl moiety, an octadecadienyl moiety, an icosadienylmoiety, a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, and a branched alkyl group as described above (e.g., a phytanylmoiety), as well as acyl derivatives thereof (e.g., linoleoyl,linolenoyl, γ-linolenoyl, phytanoyl, etc.). In some instances, theoctadecadienyl moiety is a linoleyl moiety. In other instances, theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. Inparticular embodiments, R⁴, R⁵, and R⁶ are all linoleyl moieties,linolenyl moieties, γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula VIII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VIII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaVIII has a structure selected from the group consisting of:

In yet another aspect, the present invention provides a cationic lipidof Formula IX having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH,    -   wherein if q is 1, R¹ and R² are both methyl groups, R⁴ and R⁵        are both linoleyl moieties, and Y and Z are both 0, then the        alkylamino group is attached to one of the two carbons adjacent        to Y or Z (i.e., at the ‘4’ or ‘6’ position of the 6-membered        ring).

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In a particular embodiments, Y and Z are both oxygen(O). In other embodiments, R³ is absent when the pH is above the pK_(a)of the cationic lipid and R³ is hydrogen when the pH is below the pK_(a)of the cationic lipid such that the amino head group is protonated. Inan alternative embodiment, R³ is an optionally substituted C₁-C₄ alkylto provide a quaternary amine. In further embodiments, R⁴ and R⁵ areindependently an optionally substituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ orC₁₄-C₂₂ acyl.

In other embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linolenyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

The alkylamino head group of Formula IX may be attached to the ‘4’ or‘5’ position of the 6-membered ring as shown below in an exemplaryembodiment wherein R¹ and R² are both methyl groups:

In further embodiments, the 6-membered ring of Formula IX may besubstituted with 1, 2, 3, 4, or 5 independently selected C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, or hydroxyl substituents.In one particular embodiment, the 6-membered ring is substituted with 1,2, 3, 4, or 5 independently selected C₁-C₄ alkyl (e.g., methyl, ethyl,propyl, or butyl) substituents. An exemplary embodiment of a cationiclipid of Formula IX having a substituted 6-membered ring (methyl groupattached to the ‘4’ position) and wherein R¹ and R² are both methylgroups is shown below:

In particular embodiments, the cationic lipids of Formula IX may besynthesized using 2-hydroxymethyl-1,4-butanediol and 1,3,5-pentanetriol(or 3-methyl-1,3,5-pentanetriol) as starting materials.

In some embodiments, the cationic lipid of Formula IX forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula IX is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula IXhas the structure:

In still yet another aspect, the present invention provides a cationiclipid of Formula X having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least one site of unsaturation in the trans (E)        configuration;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, at least one of R⁴ and R⁵ further comprises one,two, three, four, five, six, or more sites of unsaturation in the cisand/or trans configuration. In some instances, R⁴ and R⁵ areindependently selected from any of the substituted or unsubstitutedalkyl or acyl groups described herein, wherein at least one or both ofR⁴ and R⁵ comprises at least one, two, three, four, five, or six sitesof unsaturation in the trans configuration. In one particularembodiment, R⁴ and R⁵ independently comprise a backbone of from about 12to about 22 carbon atoms (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22 carbon atoms), and one or both of R⁴ and R⁵ independently compriseat least one, two, three, four, five, or six sites of unsaturation inthe trans configuration. In some preferred embodiments, at least one ofR⁴ and R⁵ comprises an (E)-heptadeceyl moiety. In other preferredembodiments, R⁴ and R⁵ are both (E)-8-heptadeceyl moieties.

In some embodiments, the cationic lipid of Formula X forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula X is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula Xhas the structure:

In another aspect, the present invention provides a cationic lipid ofFormula XI having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are joined to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,        C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and are independently        O, S, or NH.

In some embodiments, R¹ and R² are joined to form a heterocyclic ring of5 carbon atoms and 1 nitrogen atom. In certain instances, theheterocyclic ring is substituted with a substituent such as a hydroxylgroup at the ortho, meta, and/or para positions. In a preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula XI forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XI is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula XIhas the structure:

In yet another aspect, the present invention provides a cationic lipidof Formula XII having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        a substituted C₁₂-C₂₄ alkyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In one particularembodiment, n is 1. In another particular embodiment, n is 2. In otherembodiments, R³ is absent when the pH is above the pK_(a), of thecationic lipid and R³ is hydrogen when the pH is below the pK_(a) of thecationic lipid such that the amino head group is protonated. In analternative embodiment, R³ is an optionally substituted C₁-C₄ alkyl toprovide a quaternary amine.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.In particular embodiments, R⁴ and R⁵ are both phytanyl moieties.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety. In particular embodiments, R⁴ and R⁵ are both phytanoylmoieties.

In some embodiments, the cationic lipid of Formula XII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaXII has a structure selected from the group consisting of:

The compounds of the invention may be prepared by known organicsynthesis techniques, including the methods described in the Examples.In some embodiments, the synthesis of the cationic lipids of theinvention may require the use of protecting groups. Protecting groupmethodology is well known to those skilled in the art (see, e.g.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et. al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates the unwanted reactivity of a functional group. A protectinggroup can be added to a functional group to mask its reactivity duringcertain reactions and then removed to reveal the original functionalgroup. In certain instances, an “alcohol protecting group” is used. An“alcohol protecting group” is any group which decreases or eliminatesthe unwanted reactivity of an alcohol functional group. Protectinggroups can be added and removed using techniques well known in the art.

In certain embodiments, the cationic lipids of the present inventionhave at least one protonatable or deprotonatable group, such that thelipid is positively charged at a pH at or below physiological pH (e.g.,pH 7.4), and neutral at a second pH, preferably at or abovephysiological pH. It will be understood by one of ordinary skill in theart that the addition or removal of protons as a function of pH is anequilibrium process, and that the reference to a charged or a neutrallipid refers to the nature of the predominant species and does notrequire that all of the lipid be present in the charged or neutral form.Lipids that have more than one protonatable or deprotonatable group, orwhich are zwiterrionic, are not excluded from use in the invention.

In certain other embodiments, protonatable lipids according to theinvention have a pK_(a) of the protonatable group in the range of about4 to about 11. Most preferred is a pK_(a) of about 4 to about 7, becausethese lipids will be cationic at a lower pH formulation stage, whileparticles will be largely (though not completely) surface neutralized atphysiological pH of around pH 7.4. One of the benefits of this pK_(a) isthat at least some nucleic acid associated with the outside surface ofthe particle will lose its electrostatic interaction at physiological pHand be removed by simple dialysis; thus greatly reducing the particle'ssusceptibility to clearance.

IV. Active Agents

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, organ,or subject. Such effects may be, e.g., biological, physiological, and/orcosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., siRNA, Dicer-substratedsRNA, shRNA, aiRNA, and/or miRNA), antisense oligonucleotides,plasmids, ribozymes, immunostimulatory oligonucleotides, and mixturesthereof. Examples of peptides or polypeptides include, withoutlimitation, antibodies (e.g., polyclonal antibodies, monoclonalantibodies, antibody fragments; humanized antibodies, recombinantantibodies, recombinant human antibodies, and/or Primatized™antibodies), cytokines, growth factors, apoptotic factors,differentiation-inducing factors, cell-surface receptors and theirligands, hormones, and mixtures thereof. Examples of small moleculesinclude, but are not limited to, small organic molecules or compoundssuch as any conventional agent or drug known to those of skill in theart.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

A. Nucleic Acids

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., SNALP). In some embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” includes any oligonucleotide or polynucleotide, with fragmentscontaining up to 60 nucleotides generally termed oligonucleotides, andlonger fragments termed polynucleotides. In particular embodiments,oligonucletoides of the invention are from about 15 to about 60nucleotides in length. Nucleic acid may be administered alone in thelipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles of the invention comprisingpeptides, polypeptides, or small molecules such as conventional drugs.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a nucleic acid-lipid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA are described herein and include, e.g., structuralgenes, genes including control and termination regions, andself-replicating systems such as viral or plasmid DNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as Dicer-substrate dsRNA, shRNA, aiRNA, andpre-miRNA. Single-stranded nucleic acids include, e.g., antisenseoligonucleotides, ribozymes, mature miRNA, and triplex-formingoligonucleotides. In further embodiments, the nucleic acids aredouble-stranded DNA. Examples of double-stranded DNA include, e.g.,DNA-DNA hybrids comprising a DNA sense strand and a DNA antisense strandas described in PCT Publicaiton No. WO 2004/104199, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressiontherefrom, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

1. siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest. Each strand of the siRNA duplex is typically about 15 to about60 nucleotides in length, preferably about 15 to about 30 nucleotides inlength. In certain embodiments, the siRNA comprises at least onemodified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. The modifiednucleotides can be present in one strand (i.e., sense or antisense) orboth strands of the siRNA. In some preferred embodiments, one or more ofthe uridine and/or guanosine nucleotides are modified (e.g.,2′OMe-modified) in one strand (i.e., sense or antisense) or both strandsof the siRNA. In these embodiments, the modified siRNA can furthercomprise one or more modified (e.g., 2′OMe-modified) adenosine and/ormodified (e.g., 2′OMe-modified) cytosine nucleotides. In other preferredembodiments, only uridine and/or guanosine nucleotides are modified(e.g., 2′OMe-modified) in one strand (i.e., sense or antisense) or bothstrands of the siRNA. The siRNA sequences may have overhangs (e.g., 3′or 5′ overhangs as described in Elbashir et al., Genes Dev., 15:188(2001) or Nykänen et al., Cell, 107:309 (2001)), or may lack overhangs(i.e., have blunt ends).

In particular embodiments, the selective incorporation of modifiednucleotides such as 2′OMe uridine and/or guanosine nucleotides into thedouble-stranded region of either or both strands of the siRNA reduces orcompletely abrogates the immune response to that siRNA molecule. Incertain instances, the immunostimulatory properties of specific siRNAsequences and their ability to silence gene expression can be balancedor optimized by the introduction of minimal and selective 2′OMemodifications within the double-stranded region of the siRNA duplex.This can be achieved at therapeutically viable siRNA doses withoutcytokine induction, toxicity, and off-target effects associated with theuse of unmodified siRNA.

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. Incertain other embodiments, some or all of the modified nucleotides inthe double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more nucleotides apart from each other. In one preferredembodiment, none of the modified nucleotides in the double-strandedregion of the siRNA are adjacent to each other (e.g., there is a gap ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides betweeneach modified nucleotide).

In some embodiments, less than about 50% (e.g., less than about 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%,preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of thenucleotides in the double-stranded region of the siRNA comprise modified(e.g., 2′OMe) nucleotides. In one aspect of these embodiments, less thanabout 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, lessthan about 50% of the nucleotides in the double-stranded region of thesiRNA comprise 2′OMe nucleotides, wherein the siRNA comprises 2′OMenucleotides in both strands of the siRNA, wherein the siRNA comprises atleast one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridinenucleotide, and wherein the siRNA does not comprise 2′OMe-cytosinenucleotides in the double-stranded region. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the 2′OMe nucleotides in the double-stranded region are notadjacent to each other.

In other embodiments, from about 1% to about 50% (e.g., from about5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%,45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%,40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%,25%-37%, 25%-36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%,27%-37%, 27%-36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%,29%-37%, 29%-36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%,31%-38%, 31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%,5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%, 23%-35%, 24%-35%,25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%, 30%-35%, 31%-35%, 32%-35%,33%-35%, 34%-35%, 30%-34%, 31%-34%, 32%-34%, 33%-34%, 30%-33%, 31%-33%,32%-33%, 30%-32%, 31%-32%, 25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%;26%-33%, 26%-32%, 26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%,28%-33%, 28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%, 21%-30%,22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%, 25%-27%, 25%-26%,26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%, 27%-29%, 27%-28%, 28%-30%,28%-29%, 29%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-29%, 20%-28%, 20%-27%,20%-26%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%)of the nucleotides in the double-stranded region of the siRNA comprisemodified nucleotides. In one aspect of these embodiments, from about 1%to about 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, fromabout 1% to about 50% of the nucleotides in the double-stranded regionof the siRNA comprise 2′OMe nucleotides, wherein the siRNA comprises2′OMe nucleotides in both strands of the siRNA, wherein the siRNAcomprises at least one 2′OMe-guanosine nucleotide and at least one2′OMe-uridine nucleotide, and wherein the siRNA does not comprise2′OMe-cytosine nucleotides in the double-stranded region. In anotheraspect of these embodiments, from about 1% to about 50% of thenucleotides in the double-stranded region of the siRNA comprise 2′OMenucleotides, wherein the siRNA comprises 2′OMe nucleotides in bothstrands of the modified siRNA, wherein the siRNA comprises 2′OMenucleotides selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, andmixtures thereof, and wherein the 2′OMe nucleotides in thedouble-stranded region are not adjacent to each other.

In certain embodiments, the siRNA component of the nucleic acid-lipidparticles of the present invention (e.g., SNALP) comprises an asymmetricsiRNA duplex as described in PCT Publication No. WO 2004/078941, whichcomprises a double-stranded region consisting of a DNA sense strand andan RNA antisense strand (e.g., a DNA-RNA hybrid), wherein a blockingagent is located on the siRNA duplex. In some instances, the asymmetricsiRNA duplex can be chemically modified as described herein. Othernon-limiting examples of asymmetric siRNA duplexes are described in PCTPublication No. WO 2006/074108, which discloses self-protectedoligonucleotides comprising a region having a sequence complementary toone, two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs) and one or more self-complementary regions. Yetother non-limiting examples of asymmetric siRNA duplexes are describedin PCT Publication No. WO 2009/076321, which discloses self-formingasymmetric precursor polynucleotides comprising a targeting regioncomprising a polynucleotide sequence complementary to a region of one,two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Additional ranges, percentages, and patterns of modifications that maybe introduced into siRNA are described in U.S. Patent Publication No.20070135372, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

a) Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

As a non-limiting example, the nucleotide sequence 3′ of the AUG startcodon of a transcript from the target gene of interest may be scannedfor dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N═C, G,or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Thenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences (i.e., a target sequence or a sense strandsequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or morenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences. In some embodiments, the dinucleotidesequence is an AA or NA sequence and the 19 nucleotides immediately 3′to the AA or NA dinucleotide are identified as potential siRNAsequences. siRNA sequences are usually spaced at different positionsalong the length of the target gene. To further enhance silencingefficiency of the siRNA sequences, potential siRNA sequences may beanalyzed to identify sites that do not contain regions of homology toother coding sequences, e.g., in the target cell or organism. Forexample, a suitable siRNA sequence of about 21 base pairs typically willnot have more than 16-17 contiguous base pairs of homology to codingsequences in the target cell or organism. If the siRNA sequences are tobe expressed from an RNA Pol III promoter, siRNA sequences lacking morethan 4 contiguous A's or T's are selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g.,http://ihome.ust.hk/˜bokcmho/siRNAJsiRNA.html. One of skill in the artwill appreciate that sequences with one or more of the foregoingcharacteristics may be selected for further analysis and testing aspotential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available at http://mfold.burnet.edu.au/rna_form) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′,5′-UGU-3′,5′-GUGU-3′,5′-UGUGU-3′, etc.) can alsoprovide an indication of whether the sequence may be immunostimulatory.Once an siRNA molecule is found to be immunostimulatory, it can then bemodified to decrease its immunostimulatory properties as describedherein. As a non-limiting example, an siRNA sequence can be contactedwith a mammalian responder cell under conditions such that the cellproduces a detectable immune response to determine whether the siRNA isan immunostimulatory or a non-immunostimulatory siRNA. The mammalianresponder cell may be from a naïve mammal (i.e., a mammal that has notpreviously been in contact with the gene product of the siRNA sequence).The mammalian responder cell may be, e.g., a peripheral bloodmononuclear cell (PBMC), a macrophage, and the like. The detectableimmune response may comprise production of a cytokine or growth factorsuch as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-8, IL-12, or acombination thereof. An siRNA molecule identified as beingimmunostimulatory can then be modified to decrease its immunostimulatoryproperties by replacing at least one of the nucleotides on the senseand/or antisense strand with modified nucleotides. For example, lessthan about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) ofthe nucleotides in the double-stranded region of the siRNA duplex can bereplaced with modified nucleotides such as 2′OMe nucleotides. Themodified siRNA can then be contacted with a mammalian responder cell asdescribed above to confirm that its immunostimulatory properties havebeen reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α(PBL Biomedical;Piscataway, N.J.); human IL-6 and TNF-α(eBioscience; San Diego, Calif.);and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

b) Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

c) Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA moleculestrikes a balance between reduction or abrogation of theimmunostimulatory properties of the siRNA and retention of RNAiactivity. As a non-limiting example, an siRNA molecule that targets agene of interest can be minimally modified (e.g., less than about 30%,25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/orguanosine nucleotides within the siRNA duplex to eliminate the immuneresponse generated by the siRNA while retaining its capability tosilence target gene expression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides,2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl)nucleotides, and 2′-azido nucleotides. In certain instances, the siRNAmolecules described herein include one or more G-clamp nucleotides. AG-clamp nucleotide refers to a modified cytosine analog wherein themodifications confer the ability to hydrogen bond both Watson-Crick andHoogsteen faces of a complementary guanine nucleotide within a duplex(see, e.g., Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). Inaddition, nucleotides having a nucleotide base analog such as, forexample, C-phenyl, C-naphthyl, other aromatic derivatives, inosine,azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes,Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into siRNAmolecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3%2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3% phosphoramidate,5% phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e. resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides, modified (e.g.,2′OMe) and/or unmodified uridine ribonucleotides, and/or any othercombination of modified (e.g., 2′OMe) and unmodified nucleotides.

Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into siRNA molecules are described,e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos.20040192626, 20050282188, and 20070135372, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

d) Target Genes

The siRNA component of the nucleic acid-lipid particles described hereincan be used to downregulate or silence the translation (i.e.,expression) of a gene of interest. Genes of interest include, but arenot limited to, genes associated with viral infection and survival,genes associated with metabolic diseases and disorders (e.g., liverdiseases and disorders), genes associated with tumorigenesis or celltransformation (e.g., cancer), angiogenic genes, immunomodulator genessuch as those associated with inflammatory and autoimmune responses,receptor ligand genes, and genes associated with neurodegenerativedisorders.

In particular embodiments, the present invention provides a cocktail oftwo, three, four, five, six, seven, eight, nine, ten, or more siRNAmolecules that silences the expression of multiple genes of interest. Insome embodiments, the cocktail of siRNA molecules is fully encapsulatedin a lipid particle such as a nucleic acid-lipid particle (e.g., SNALP).The siRNA molecules may be co-encapsulated in the same lipid particle,or each siRNA species present in the cocktail may be formulated inseparate particles.

Genes associated with viral infection and survival include thoseexpressed by a host (e.g., a host factor such as tissue factor (TF)) ora virus in order to bind, enter, and replicate in a cell. Of particularinterest are viral sequences associated with chronic viral diseases.Viral sequences of particular interest include sequences of Filovirusessuch as Ebola virus and Marburg virus (see, e.g., Geisbert et al., J.Infect. Dis., 193:1650-1657 (2006)); Arenaviruses such as Lassa virus,Junin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeieret al., Arenaviridae: the viruses and their replication, In: FIELDSVIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia,(2001)); Influenza viruses such as Influenza A, B, and C viruses, (see,e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002); and Neumannet al., J Gen Virol., 83:2635-2662 (2002)); Hepatitis viruses (see,e.g., Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBORep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilsonet al., Proc. Natl. Acad. Sci. USA, 100:2783 (2003); Kapadia et al.,Proc. Natl. Acad. Sci. USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipeet al. (eds.), 4th ed., Lippincott-Raven, Philadelphia (2001)); HumanImmunodeficiency Virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003);Song et al., J. Virol., 77:7174 (2003); Stephenson, JAMA, 289:1494(2003); Qin et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003)); Herpesviruses (Jia et al., J. Virol., 77:3301 (2003)); and Human PapillomaViruses (HPV) (Hall et al., J. Virol., 77:6066 (2003); Jiang et al.,Oncogene, 21:6041 (2002)).

Exemplary Filovirus nucleic acid sequences that can be silenced include,but are not limited to, nucleic acid sequences encoding structuralproteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein(L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein(GP), VP24). Complete genome sequences for Ebola virus are set forth in,e.g., Genbank Accession Nos. NC_(—)002549; AY769362; NC_(—)006432;NC_(—)004161; AY729654; AY354458; AY142960; AB050936; AF522874;AF499101; AF272001; and AF086833. Ebola virus VP24 sequences are setforth in, e.g., Genbank Accession Nos. U77385 and AY058897. Ebola virusL-pol sequences are set forth in, e.g., Genbank Accession No. X67110.Ebola virus VP40 sequences are set forth in, e.g., Genbank Accession No.AY058896. Ebola virus NP sequences are set forth in, e.g., GenbankAccession No. AY058895. Ebola virus GP sequences are set forth in, e.g.,Genbank Accession No. AY058898; Sanchez et al., Virus Res., 29:215-240(1993); Will et al., J. Virol., 67:1203-1210 (1993); Volchkov et al.,FEBS Lett., 305:181-184 (1992); and U.S. Pat. No. 6,713,069. AdditionalEbola virus sequences are set forth in, e.g., Genbank Accession Nos.L11365 and X61274. Complete genome sequences for Marburg virus are setforth in, e.g., Genbank Accession Nos. NC_(—)001608; AY430365; AY430366;and AY358025. Marburg virus GP sequences are set forth in, e.g., GenbankAccession Nos. AF005734; AF005733; and AF005732. Marburg virus VP35sequences are set forth in, e.g., Genbank Accession Nos. AF005731 andAF005730. Additional Marburg virus sequences are set forth in, e.g.,Genbank Accession Nos. X64406; Z29337; AF005735; and Z12132.Non-limiting examples of siRNA molecules targeting Ebola virus andMarburg virus nucleic acid sequences include those described in U.S.Patent Publication No. 20070135370 and U.S. Provisional Application No.61/286,741, filed Dec. 15, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Exemplary Arenavirus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), glycoprotein (GP), L-polymerase (L), and Z protein(Z). Complete genome sequences for Lassa virus are set forth in, e.g.,Genbank Accession Nos. NC_(—)004296 (LASV segment S) and NC_(—)004297(LASV segment L). Non-limiting examples of siRNA molecules targetingLassa virus nucleic acid sequences include those described in U.S.Provisional Application No. 61/319,855, filed Mar. 31, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Exemplary host nucleic acid sequences that can be silenced include, butare not limited to, nucleic acid sequences encoding host factors such astissue factor (TF) that are known to play a role in the pathogenisis ofhemorrhagic fever viruses. The mRNA sequence of TF is set forth inGenbank Accession No. NM_(—)001993. Those of skill in the art willappreciate that TF is also known as F3, coagulation factor III,thromboplastin, and CD142. Non-limiting examples of siRNA moleculestargeting TF nucleic acid sequences include those described in U.S.Provisional Application No. 61/319,855, filed Mar. 31, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Exemplary Influenza virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins(NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), andhaemagglutinin (HA). Influenza A NP sequences are set forth in, e.g.,Genbank Accession Nos. NC_(—)004522; AY818138; AB166863; AB188817;AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493;AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507;AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140.Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos.AY818132; AY790280; AY646171; AY818132;AY818133; AY646179; AY818134;AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611;AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615;AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995;and AY724786. Non-limiting examples of siRNA molecules targetingInfluenza virus nucleic acid sequences include those described in U.S.Patent Publication No. 20070218122, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary hepatitis virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences involved intranscription and translation (e.g., En1, En2, X, P) and nucleic acidsequences encoding structural proteins (e.g., core proteins including Cand C-related proteins, capsid and envelope proteins including S, M,and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY,supra). Exemplary Hepatits C virus (HCV) nucleic acid sequences that canbe silenced include, but are not limited to, the 5′-untranslated region(5′-UTR), the 3′-untranslated region (3′-UTR), the polyproteintranslation initiation codon region, the internal ribosome entry site(IRES) sequence, and/or nucleic acid sequences encoding the coreprotein, the E1 protein, the E2 protein, the p7 protein, the NS2protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein,the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCVgenome sequences are set forth in, e.g., Genbank Accession Nos.NC_(—)004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_(—)009823(HCV genotype 2), NC_(—)009824 (HCV genotype 3), NC_(—)009825 (HCVgenotype 4), NC_(—)009826 (HCV genotype 5), and NC_(—)009827 (HCVgenotype 6). Hepatitis A virus nucleic acid sequences are set forth in,e.g., Genbank Accession No. NC_(—)001489; Hepatitis B virus nucleic acidsequences are set forth in, e.g., Genbank Accession No. NC_(—)003977;Hepatitis D virus nucleic acid sequence are set forth in, e.g., GenbankAccession No. NC_(—)001653; Hepatitis E virus nucleic acid sequences areset forth in, e.g., Genbank Accession No. NC_(—)001434; and Hepatitis Gvirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_(—)001710. Silencing of sequences that encode genes associatedwith viral infection and survival can conveniently be used incombination with the administration of conventional agents used to treatthe viral condition. Non-limiting examples of siRNA molecules targetinghepatitis virus nucleic acid sequences include those described in U.S.Patent Publication Nos. 20060281175, 20050058982, and 20070149470; U.S.Pat. No. 7,348,314; and PCT Application No. PCT/CA2010/000444, entitled“Compositions and Methods for Silencing Hepatitis C Virus Expression,”filed Mar. 19, 2010, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, but are not limited to, genes expressed in dyslipidemia, suchas, e.g., apolipoprotein B (APOB) (Genbank Accession No. NM_(—)000384),apolipoprotein CIII (APOC3) (Genbank Accession Nos. NM_(—)000040 andNG_(—)008949 REGION: 5001.8164), apolipoprotein E (APOE) (GenbankAccession Nos. NM_(—)000041 and NG_(—)007084 REGION: 5001.8612),proprotein convertase subtilisin/kexin type 9 (PCSK9) (Genbank AccessionNo. NM_(—)174936), diacylglycerol O-acyltransferase type 1 (DGAT1)(Genbank Accession No. NM_(—)012079), diacylglyerol O-acyltransferasetype 2 (DGAT2) (Genbank Accession No. NM_(—)032564), liver X receptorssuch as LXRα and LXRβ (Genback Accession No. NM_(—)007121), farnesoid Xreceptors (FXR) (Genbank Accession No. NM_(—)005123), sterol-regulatoryelement binding protein (SREBP), site-1 protease (SIP),3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-Areductase); and genes expressed in diabetes, such as, e.g., glucose6-phosphatase (see, e.g., Forman et al., Cell, 81:687 (1995); Seol etal., Mol. Endocrinol., 9:72 (1995), Zavacki et al., Proc. Natl. Acad.Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996);Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et al.,Genes Dev., 9:1033-1045 (1995); Lehmann et al., J. Biol. Chem.,272:3137-3140 (1997); Janowski et al., Nature, 383:728-731 (1996); andPeet et al., Cell, 93:693-704 (1998)).

One of skill in the art will appreciate that genes associated withmetabolic diseases and disorders (e.g., diseases and disorders in whichthe liver is a target and liver diseases and disorders) include genesthat are expressed in the liver itself as well as and genes expressed inother organs and tissues. Silencing of sequences that encode genesassociated with metabolic diseases and disorders can conveniently beused in combination with the administration of conventional agents usedto treat the disease or disorder. Non-limiting examples of siRNAmolecules targeting the APOB gene include those described in U.S. PatentPublication Nos. 20060134189, 20060105976, and 20070135372, and PCTPublication No. WO 04/091515, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.Non-limiting examples of siRNA molecules targeting the APOC3 geneinclude those described in PCT Application No. PCT/CA2010/000120, filedJan. 26, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes. Non-limiting examples ofsiRNA molecules targeting the PCSK9 gene include those described in U.S.Patent Publication Nos. 20070173473, 20080113930, and 20080306015, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes. Exemplary siRNA molecules targeting the DGAT1gene may be designed using the antisense compounds described in U.S.Patent Publication No. 20040185559, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. ExemplarysiRNA molecules targeting the DGAT2 gene may be designed using theantisense compounds described in U.S. Patent Publication No.20050043524, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

Genes associated with tumorigenesis or cell transformation (e.g., canceror other neoplasia) include, for example, genes involved in p53ubiquitination, c-Jun ubiquitination, histone deacetylation, cell cycleregulation, transcriptional regulation, and combinations thereof.Non-limiting examples of gene sequences associated with tumorigenesis orcell transformation include serine/threonine kinases such as polo-likekinase 1 (PLK-1) (Genbank Accession No. NM_(—)005030; Barr et al., Nat.Rev. Mol. Cell. Biol., 5:429-440 (2004)) and cyclin-dependent kinase 4(CDK4) (Genbank Accession No. NM_(—)000075); ubiquitin ligases such asCOP1 (RFWD2; Genbank Accession Nos. NM_(—)022457 and NM_(—)001001740)and ring-box 1 (RBX1) (ROC1; Genbank Accession No. NM_(—)014248);tyrosine kinases such as WEE1 (Genbank Accession Nos. NM_(—)003390 andNM_(—)001143976); mitotic kinesins such as Eg5 (KSP, KIF11; GenbankAccession No. NM_(—)004523); transcription factors such as forkhead boxM1 (FOXM1) (Genbank Accession Nos. NM_(—)202002, NM_(—)021953, andNM_(—)202003) and RAM2 (R1 or CDCA7L; Genbank Accession Nos.NM_(—)018719, NM_(—)001127370, and NM_(—)001127371); inhibitors ofapoptosis such as XIAP (Genbank Accession No. NM_(—)001167); COP9signalosome subunits such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; GenbankAccession No. NM_(—)006837); CSN6, CSN7A, CSN7B, and CSN8; and histonedeacetylases such as HDAC1, HDAC2 (Genbank Accession No. NM_(—)001527),HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc.

Non-limiting examples of siRNA molecules targeting the PLK-1 geneinclude those described in U.S. Patent Publication Nos. 20050107316 and20070265438; and PCT Publication No. WO 09/082,817, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. Non-limiting examples of siRNA molecules targeting the Eg5 andXIAP genes include those described in U.S. Patent Publication No.20090149403, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Non-limiting examples of siRNAmolecules targeting the CSN5 gene include those described in PCTPublication No. WO 09/129,319, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Non-limitingexamples of siRNA molecules targeting the COP1, CSN5, RBX1, HDAC2, CDK4,WEE1, FOXM1, and RAM2 genes include those described in U.S. ProvisionalApplication No. 61/245,143, filed Sep. 23, 2009, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Additional examples of gene sequences associated with tumorigenesis orcell transformation include translocation sequences such as MLL fusiongenes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al.,Blood, 101:1566 (2003)), TEL-AMLI, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2,AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));overexpressed sequences such as multidrug resistance genes (Nieth etal., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515 (2003)),cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev.,16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase genes (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR/ErbB1(Genbank Accession Nos. NM_(—)005228, NM_(—)201282, NM_(—)201283, andNM_(—)201284; see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003)),ErbB2/HER-2 (Genbank Accession Nos. NM_(—)004448 and NM_(—)001005862),ErbB3 (Genbank Accession Nos. NM_(—)001982 and NM_(—)001005915), andErbB4 (Genbank Accession Nos. NM_(—)005235 and NM_(—)001042599)), andmutated sequences such as RAS (Tuschl and Borkhardt, Mol. Interventions,2:158 (2002)). Non-limiting examples of siRNA molecules targeting theEGFR gene include those described in U.S. Patent Publication No.20090149403, the disclosure of which is herein incorporated by referencein its entirety for all purposes. siRNA molecules that target VEGFRgenes are set forth in, e.g., GB 2396864; U.S. Patent Publication No.20040142895; and CA 2456444, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Silencing of sequences that encode DNA repair enzymes find use incombination with the administration of chemotherapeutic agents (Colliset al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associatedwith tumor migration are also target sequences of interest, for example,integrins, selectins, and metalloproteinases. The foregoing examples arenot exclusive. Those of skill in the art will understand that any wholeor partial gene sequence that facilitates or promotes tumorigenesis orcell transformation, tumor growth, or tumor migration can be included asa template sequence.

Angiogenic genes are able to promote the formation of new vessels.Angiogenic genes of particular interest include, but are not limited to,vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis.,9:210 (2003)), placental growth factor (PGF), VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1), and the like. siRNA molecules that target VEGFR genes areset forth in, e.g., GB 2396864; U.S. Patent Publication No. 20040142895;and CA 2456444, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include, withoutlimitation, growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF,PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12(Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.),interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fasligand genes are also immunomodulator target sequences of interest (Songet al., Nat. Med., 9:347 (2003)). Genes encoding secondary signalingmolecules in hematopoietic and lymphoid cells are also included in thepresent invention, for example, Tec family kinases such as Bruton'styrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

Cell receptor ligand genes include ligands that are able to bind to cellsurface receptors (e.g., cytokine receptors, growth factor receptors,receptors with tyrosine kinase activity, G-protein coupled receptors,insulin receptor, EPO receptor, etc.) to modulate (e.g., inhibit) thephysiological pathway that the receptor is involved in (e.g., cellproliferation, tumorigenesis, cell transformation, mitogenesis, etc.).Non-limiting examples of cell receptor ligand genes include cytokines(e.g., TNF-α, interferons such as IFN-α, IFN-β, and IFN-γ, interleukinssuch as IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-17, IL-23, IL-27, chemokines, etc.), growthfactors (e.g., EGF, HB-EGF, VEGF, PEDF, SDGF, bFGF, HGF, TGF-α, TGF-β,BMP1-BMP15, PDGF, IGF, NGF, β-NGF, BDNF, NT3, NT4, GDF-9, CGF, G-CSF,GM-CSF, GDF-8, EPO, TPO, etc.), insulin, glucagon, G-protein coupledreceptor ligands, etc.

Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats) find use in silencing pathogenic sequences in neurodegenerativedisorders caused by the expansion of trinucleotide repeats, such asspinobulbular muscular atrophy and Huntington's Disease (Caplen et al.,Hum. Mol. Genet., 11:175 (2002)).

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, the siRNA describedherein are also useful in research and development applications as wellas diagnostic, prophylactic, prognostic, clinical, and other healthcareapplications. As a non-limiting example, the siRNA can be used in targetvalidation studies directed at testing whether a gene of interest hasthe potential to be a therapeutic target. The siRNA can also be used intarget identification studies aimed at discovering genes as potentialtherapeutic targets.

e) Exemplary siRNA Embodiments

In some embodiments, each strand of the siRNA molecule comprises fromabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50,15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particularembodiment, the siRNA is chemically synthesized. The siRNA molecules ofthe invention are capable of silencing the expression of a targetsequence in vitro and/or in vivo.

In other embodiments, the siRNA comprises at least one modifiednucleotide. In certain embodiments, the siRNA comprises one, two, three,four, five, six, seven, eight, nine, ten, or more modified nucleotidesin the double-stranded region. In particular embodiments, less thanabout 50% (e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5%) of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides. In preferred embodiments, fromabout 1% to about 50% (e.g., from about 5%-50%, 10%-50%, 15%-50%,20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%,15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%,15%-40%, 20%-40%, 25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%,20%-35%, 25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%,10%-15%, or 5%-10%) of the nucleotides in the double-stranded region ofthe siRNA comprise modified nucleotides.

In further embodiments, the siRNA comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, e.g., 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, ormixtures thereof. In one particular embodiment, the siRNA comprises atleast one 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, ormixtures thereof. In certain instances, the siRNA does not comprise2′OMe-cytosine nucleotides. In other embodiments, the siRNA comprises ahairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in onestrand (i.e., sense or antisense) or both strands of the double-strandedregion of the siRNA molecule. Preferably, uridine and/or guanosinenucleotides are modified at selective positions in the double-strandedregion of the siRNA duplex. With regard to uridine nucleotidemodifications, at least one, two, three, four, five, six, or more of theuridine nucleotides in the sense and/or antisense strand can be amodified uridine nucleotide such as a 2′OMe-uridine nucleotide. In someembodiments, every uridine nucleotide in the sense and/or antisensestrand is a 2′OMe-uridine nucleotide. With regard to guanosinenucleotide modifications, at least one, two, three, four, five, six, ormore of the guanosine nucleotides in the sense and/or antisense strandcan be a modified guanosine nucleotide such as a 2′OMe-guanosinenucleotide. In some embodiments, every guanosine nucleotide in the senseand/or antisense strand is a 2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the SNALP deliverysystem disclosed herein).

In other embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In another embodiment, an unmodified or modified siRNA molecule iscapable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% of the expression of the target sequencerelative to a negative control (e.g., buffer only, an siRNA sequencethat targets a different gene, a scrambled siRNA sequence, etc.).

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the expression of the target sequence relative tothe corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. In certain embodiments,the 3′ overhang on the sense and/or antisense strand independentlycomprises one, two, three, four, or more modified nucleotides such as2′OMe nucleotides and/or any other modified nucleotide described hereinor known in the art.

In particular embodiments, siRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more siRNAmolecules; (b) a cationic lipid of Formula I-XII or a salt thereof; and(c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).In certain instances, the nucleic acid-lipid particle may furthercomprise a conjugated lipid that prevents aggregation of particles(e.g., PEG-DAA).

2. Dicer-Substrate dsRNA

As used herein, the term “Dicer-substrate dsRNA” or “precursor RNAimolecule” is intended to include any precursor molecule that isprocessed in vivo by Dicer to produce an active siRNA which isincorporated into the RISC complex for RNA interference of a targetgene.

In one embodiment, the Dicer-substrate dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA. According tothis embodiment, the Dicer-substrate dsRNA comprises (i) a firstoligonucleotide sequence (also termed the sense strand) that is betweenabout 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55,25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferablybetween about 25 and about 30 nucleotides in length (e.g., 25, 26, 27,28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. The second oligonucleotide sequence may bebetween about 25 and about 60 nucleotides in length (e.g., about 25-60,25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), andis preferably between about 25 and about 30 nucleotides in length (e.g.,25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a regionof one of the sequences, particularly of the antisense strand, of theDicer-substrate dsRNA has a sequence length of at least about 19nucleotides, for example, from about 19 to about 60 nucleotides (e.g.,about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and has at least one of the following properties: (i)the dsRNA is asymmetric, e.g., has a 3′-overhang on the antisensestrand; and/or (ii) the dsRNA has a modified 3′-end on the sense strandto direct orientation of Dicer binding and processing of the dsRNA to anactive siRNA. According to this latter embodiment, the sense strandcomprises from about 22 to about 28 nucleotides and the antisense strandcomprises from about 24 to about 30 nucleotides.

In one embodiment, the Dicer-substrate dsRNA has an overhang on the3′-end of the antisense strand. In another embodiment, the sense strandis modified for Dicer binding and processing by suitable modifierslocated at the 3′-end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the Dicer-substrate dsRNA has an overhangon the 3′-end of the antisense strand and the sense strand is modifiedfor Dicer processing. In another embodiment, the 5′-end of the sensestrand has a phosphate. In another embodiment, the 5′-end of theantisense strand has a phosphate. In another embodiment, the antisensestrand or the sense strand or both strands have one or more 2′-O -methyl(2′OMe) modified nucleotides. In another embodiment, the antisensestrand contains 2′OMe modified nucleotides. In another embodiment, theantisense stand contains a 3′-overhang that is comprised of 2′OMemodified nucleotides. The antisense strand could also include additional2′OMe modified nucleotides. The sense and antisense strands anneal underbiological conditions, such as the conditions found in the cytoplasm ofa cell. In addition, a region of one of the sequences, particularly ofthe antisense strand, of the Dicer-substrate dsRNA has a sequence lengthof at least about 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′-end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene. Further, in accordance with thisembodiment, the Dicer-substrate dsRNA may also have one or more of thefollowing additional properties: (a) the antisense strand has a rightshift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the right side of the molecule when compared to thetypical 21-mer); (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings; and (c) basemodifications such as locked nucleic acid(s) may be included in the5′-end of the sense strand.

In a third embodiment, the sense strand comprises from about 25 to about28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2nucleotides on the 3′-end of the sense strand are deoxyribonucleotides.The sense strand contains a phosphate at the 5′-end. The antisensestrand comprises from about 26 to about 30 nucleotides (e.g., 26, 27,28, 29, or 30 nucleotides) and contains a 3′-overhang of 1-4nucleotides. The nucleotides comprising the 3′-overhang are modifiedwith 2′OMe modified ribonucleotides. The antisense strand containsalternating 2′OMe modified nucleotides beginning at the first monomer ofthe antisense strand adjacent to the 3′-overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′-overhang. Forexample, for a 27-nucleotide antisense strand and counting the firstbase at the 5′-end of the antisense strand as position number 1, 2′OMemodifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23,25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA has thefollowing structure:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′OMe RNA, “Y” is anoverhang domain comprised of 1, 2, 3, or 4 RNA monomers that areoptionally 2′OMe RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and(ii) the dsRNA has a modified 3′-end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the sense strand comprises fromabout 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30nucleotides) and the antisense strand comprises from about 22 to about28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In oneembodiment, the Dicer-substrate dsRNA has an overhang on the 3′-end ofthe sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′-end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the dsRNA has an overhang on the 3′-endof the sense strand and the antisense strand is modified for Dicerprocessing. In one embodiment, the antisense strand has a 5′-phosphate.The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′-end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene. Further, in accordance with this embodiment, theDicer-substrate dsRNA may also have one or more of the followingadditional properties: (a) the antisense strand has a left shift fromthe typical 21-mer (i.e., the antisense strand includes nucleotides onthe left side of the molecule when compared to the typical 21-mer); and(b) the strands may not be completely complementary, i.e., the strandsmay contain simple mismatch pairings.

In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.In certain instances, this dsRNA having an asymmetric structure furthercontains 2 deoxynucleotides at the 3′-end of the sense strand in placeof two of the ribonucleotides. In certain other instances, this dsRNAhaving an asymmetric structure further contains 2′OMe modifications atpositions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand(wherein the first base at the 5′-end of the antisense strand isposition 1). In certain additional instances, this dsRNA having anasymmetric structure further contains a 3′-overhang on the antisensestrand comprising 1, 2, 3, or 4 2′OMe nucleotides (e.g., a 3′-overhangof 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

In another embodiment, Dicer-substrate dsRNAs may be designed by firstselecting an antisense strand siRNA sequence having a length of at least19 nucleotides. In some instances, the antisense siRNA is modified toinclude about 5 to about 11 ribonucleotides on the 5′-end to provide alength of about 24 to about 30 nucleotides. When the antisense strandhas a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably6 nucleotides may be added on the 5′-end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has about 22 to about 28 nucleotides. The sense strand issubstantially complementary with the antisense strand to anneal to theantisense strand under biological conditions. In one embodiment, thesense strand is synthesized to contain a modified 3′-end to direct Dicerprocessing of the antisense strand. In another embodiment, the antisensestrand of the dsRNA has a 3′-overhang. In a further embodiment, thesense strand is synthesized to contain a modified 3′-end for Dicerbinding and processing and the antisense strand of the dsRNA has a3′-overhang.

In a related embodiment, the antisense siRNA may be modified to includeabout 1 to about 9 ribonucleotides on the 5′-end to provide a length ofabout 22 to about 28 nucleotides. When the antisense strand has a lengthof 21 nucleotides, 1-7, preferably 2-5, or more preferably 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has about 24 toabout 30 nucleotides. The sense strand is substantially complementarywith the antisense strand to anneal to the antisense strand underbiological conditions. In one embodiment, the antisense strand issynthesized to contain a modified 3′-end to direct Dicer processing. Inanother embodiment, the sense strand of the dsRNA has a 3′-overhang. Ina further embodiment, the antisense strand is synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′-overhang.

Suitable Dicer-substrate dsRNA sequences can be identified, synthesized,and modified using any means known in the art for designing,synthesizing, and modifying siRNA sequences. In particular embodiments,Dicer-substrate dsRNAs are administered using a carrier system such as anucleic acid-lipid particle (e.g., SNALP). In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or moreDicer-substrate dsRNA molecules; (b) a cationic lipid of Formula I-XIIor a salt thereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE,and/or cholesterol). In certain instances, the nucleic acid-lipidparticle may further comprise a conjugated lipid that preventsaggregation of particles (e.g., PEG-DAA).

Additional embodiments related to the Dicer-substrate dsRNAs of theinvention, as well as methods of designing and synthesizing such dsRNAs,are described in U.S. Patent Publication Nos. 20050244858, 20050277610,and 20070265220, and U.S. application Ser. No. 12/794,701, filed Jun. 4,2010, the disclosures of which are herein incorporated by reference intheir entirety for all purposes.

3. shRNA

A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a shortRNA sequence that makes a tight hairpin turn that can be used to silencegene expression via RNA interference. The shRNAs of the invention may bechemically synthesized or transcribed from a transcriptional cassette ina DNA plasmid. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC).

The shRNAs of the invention are typically about 15-60, 15-50, or 15-40(duplex) nucleotides in length, more typically about 15-30, 15-25, or19-25 (duplex) nucleotides in length, and are preferably about 20-24,21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementarysequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30,15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or21-23 nucleotides in length, and the double-stranded shRNA is about15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNAduplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides orabout 2 to about 3 nucleotides on the antisense strand and/or5′-phosphate termini on the sense strand. In some embodiments, the shRNAcomprises a sense strand and/or antisense strand sequence of from about15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferablyfrom about 19 to about 40 nucleotides in length (e.g., about 19-40,19-35, 19-30, or 19-25 nucleotides in length), more preferably fromabout 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In preferred embodiments, the sense and antisensestrands of the shRNA are linked by a loop structure comprising fromabout 1 to about 25 nucleotides, from about 2 to about 20 nucleotides,from about 4 to about 15 nucleotides, from about 5 to about 12nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Additional shRNA sequences include, but are not limited to, asymmetricshRNA precursor polynucleotides such as those described in PCTPublication Nos. WO 2006/074108 and WO 2009/076321, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. For example, PCT Publication No. WO 2006/074108 disclosesself-protected oligonucleotides comprising a region having a sequencecomplementary to one, two, three, or more same or different target mRNAsequences (e.g., multivalent shRNAs) and one or more self-complementaryregions. Similarly, PCT Publication No. WO 2009/076321 disclosesself-forming asymmetric precursor polynucleotides comprising a targetingregion comprising a polynucleotide sequence complementary to a region ofone, two, three, or more same or different target mRNA sequences (e.g.,multivalent shRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions.

Suitable shRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. In particular embodiments, shRNAs areadministered using a carrier system such as a nucleic acid-lipidparticle (e.g., SNALP). In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more shRNA molecules; (b) acationic lipid of Formula I-XII or a salt thereof; and (c) anon-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). Incertain instances, the nucleic acid-lipid particle may further comprisea conjugated lipid that prevents aggregation of particles (e.g.,PEG-DAA).

Additional embodiments related to the shRNAs of the invention, as wellas methods of designing and synthesizing such shRNAs, are described inU.S. patent application Ser. No. 12/794,701, filed Jun. 4, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

4. aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein.

In particular embodiments, aiRNAs are administered using a carriersystem such as a nucleic acid-lipid particle (e.g., SNALP). In apreferred embodiment, the nucleic acid-lipid particle comprises: (a) oneor more aiRNA molecules; (b) a cationic lipid of Formula I-XII or a saltthereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/orcholesterol). In certain instances, the nucleic acid-lipid particle mayfurther comprise a conjugated lipid that prevents aggregation ofparticles (e.g., PEG-DAA).

Suitable aiRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. Additional embodiments related to the aiRNAmolecules of the invention are described in U.S. Patent Publication No.20090291131 and PCT Publication No. WO 09/127,060, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

5. miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule miRNA are first transcribed as primary transcripts or pri-miRNAwith a cap and poly-A tail and processed to short, ˜70-nucleotidestem-loop structures known as pre-miRNA in the cell nucleus. Thisprocessing is performed in animals by a protein complex known as theMicroprocessor complex, consisting of the nuclease Drosha and thedouble-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In particular embodiments, miRNAs are administered using a carriersystem such as a nucleic acid-lipid particle (e.g., SNALP). In apreferred embodiment, the nucleic acid-lipid particle comprises: (a) oneor more miRNA molecules; (b) a cationic lipid of Formula I-XII or a saltthereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/orcholesterol). In certain instances, the nucleic acid-lipid particle mayfurther comprise a conjugated lipid that prevents aggregation ofparticles (e.g., PEG-DAA).

In other embodiments, one or more agents that block the activity of anmiRNA targeting an mRNA of interest are administered using a lipidparticle of the invention (e.g., a nucleic acid-lipid particle such asSNALP). Examples of blocking agents include, but are not limited to,steric blocking oligonucleotides, locked nucleic acid oligonucleotides,and Morpholino oligonucleotides. Such blocking agents may bind directlyto the miRNA or to the miRNA binding site on the target mRNA.

Additional embodiments related to the miRNA molecules of the inventionare described in U.S. Patent Publication No. 20090291131 and PCTPublication No. WO 09/127060, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

6. Antisense Oligonucleotides

In one embodiment, the nucleic acid is an antisense oligonucleotidedirected to a target gene or sequence of interest. The terms “antisenseoligonucleotide” or “antisense” include oligonucleotides that arecomplementary to a targeted polynucleotide sequence. Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Antisense RNA oligonucleotides prevent thetranslation of complementary RNA strands by binding to the RNA.Antisense DNA oligonucleotides can be used to target a specific,complementary (coding or non-coding) RNA. If binding occurs, thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides comprise from about 10 to about60 nucleotides, more preferably from about 15 to about 30 nucleotides.The term also encompasses antisense oligonucleotides that may not beexactly complementary to the desired target gene. Thus, the inventioncan be utilized in instances where non-target specific-activities arefound with antisense, or where an antisense sequence containing one ormore mismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(see, U.S. Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et al.,Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun.,1:225-32 (1989); Peris et al., Brain Res Mol Brain Res., 15; 57:310-20(1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Moreover, antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317;and 5,783,683). The disclosures of these references are hereinincorporated by reference in their entirety for all purposes.

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

7. Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (see, Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92(1987); and Forster et al., Cell, 49:211-20 (1987)). For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (see, Cech et al., Cell,27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAmolecules are known presently. Each can catalyze the hydrolysis of RNAphosphodiester bonds in trans (and thus can cleave other RNA molecules)under physiological conditions. In general, enzymatic nucleic acids actby first binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, hepatitis 5 virus, group I intron or RNaseP RNA (in associationwith an RNA guide sequence), or Neurospora VS RNA motif, for example.Specific examples of hammerhead motifs are described in, e.g., Rossi etal., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifsare described in, e.g., EP 0360257, Hampel et al., Biochemistry,28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);and U.S. Pat. No. 5,631,359. An example of the hepatitis 5 virus motifis described in, e.g., Perrotta et al., Biochemistry, 31:11843-52(1992). An example of the RNaseP motif is described in, e.g.,Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of theNeurospora VS RNA ribozyme motif is described in, e.g., Saville et al.,Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA,88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Anexample of the Group I intron is described in, e.g., U.S. Pat. No.4,987,071. Important characteristics of enzymatic nucleic acid moleculesused according to the invention are that they have a specific substratebinding site which is complementary to one or more of the target geneDNA or RNA regions, and that they have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus, the ribozyme constructs need not belimited to specific motifs mentioned herein. The disclosures of thesereferences are herein incorporated by reference in their entirety forall purposes.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in, e.g.,PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized to betested in vitro and/or in vivo as described therein. The disclosures ofthese PCT publications are herein incorporated by reference in theirentirety for all purposes.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162,and WO 94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, whichdescribe various chemical modifications that can be made to the sugarmoieties of enzymatic RNA molecules, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes),modifications which enhance their efficacy in cells, and removal of stemII bases to shorten RNA synthesis times and reduce chemicalrequirements.

8. Immunostimulatory Oligonucleotides

Nucleic acids associated with the lipid particles of the presentinvention may be immunostimulatory, including immunostimulatoryoligonucleotides (ISS; single- or double-stranded) capable of inducingan immune response when administered to a subject, which may be a mammalsuch as a human. ISS include, e.g., certain palindromes leading tohairpin secondary structures (see, Yamamoto et al., J. Immunol.,148:4072-6 (1992)), or CpG motifs, as well as other known ISS features(such as multi-G domains; see; PCT Publication No. WO 96/11266, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes).

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target sequence in order to provoke an immuneresponse. Thus, certain immunostimulatory nucleic acids may comprise asequence corresponding to a region of a naturally-occurring gene ormRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein the CpG dinucleotide is methylated. In an alternative embodiment, thenucleic acid comprises at least two CpG dinucleotides, wherein at leastone cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of theCpG dinucleotides comprises a methylated cytosine. Examples ofimmunostimulatory oligonucleotides suitable for use in the compositionsand methods of the present invention are described in PCT PublicationNos. WO 02/069369, WO 01/15726, and WO 09/086,558; U.S. Pat. No.6,406,705; and Raney et al., J. Pharm. Exper. Ther., 298:1185-92 (2001),the disclosures of which are herein incorporated by reference in theirentirety for all purposes. In certain embodiments, the oligonucleotidesused in the compositions and methods of the invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

B. Other Active Agents

In certain embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapaeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid such as interferingRNA.

Non-limiting examples of chemotherapy drugs include platinum-based drugs(e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin,satraplatin, etc.), alkylating agents (e.g., cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine,uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel(taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan(CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP 16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g.,gefitinib (Iressa®), sunitinib (Sutent®; SUI 1248), erlotinib (Tarceva®;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI-1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101),vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

Examples of conventional hormonal therapaeutic agents include, withoutlimitation, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and goserelin as well as othergonadotropin-releasing hormone agonists (GnRH).

Examples of conventional immunotherapeutic agents include, but are notlimited to, immunostimulants (e.g., Bacillus Calmette-Guérin (BCG),levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

Examples of conventional radiotherapeutic agents include, but are notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

Additional oncology drugs that may be used according to the inventioninclude, but are not limited to, alkeran, allopurinol, altretamine,amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU,carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosinearabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane,FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin,interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol,L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate,Pegademase, pentostatin, porfimer sodium, prednisone, rituxan,streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene,tretinoin, ATRA, valrubicin, and velban. Other examples of oncologydrugs that may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors, and camptothecins.

Non-limiting examples of lipid-lowering agents for treating a lipiddisease or disorder associated with elevated triglycerides, cholesterol,and/or glucose include statins, fibrates, ezetimibe, thiazolidinediones,niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, andmixtures thereof.

Examples of anti-viral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, entry inhibitors, famciclovir, fixed dose combinations,fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors,ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitors, interferon type III (e.g., IFN-λmolecules such as IFN-λ1, IFN-λ2, and IFN-α3), interferon type II (e.g.,IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β,IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ, interferon, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, zidovudine, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andmixtures thereof.

V. Lipid Particles

In certain aspects, the present invention provides lipid particlescomprising one or more of the cationic (amino) lipids or salts thereofdescribed herein. In some embodiments, the lipid particles of theinvention further comprise one or more non-cationic lipids. In otherembodiments, the lipid particles further comprise one or more conjugatedlipids capable of reducing or inhibiting particle aggregation. Inadditional embodiments, the lipid particles further comprise one or moreactive agents or therapeutic agents such as therapeutic nucleic acids(e.g., interfering RNA such as siRNA).

Lipid particles include, but are not limited to, lipid vesicles such asliposomes. As used herein, a lipid vesicle includes a structure havinglipid-containing membranes enclosing an aqueous interior. In particularembodiments, lipid vesicles comprising one or more of the cationiclipids described herein are used to encapsulate nucleic acids within thelipid vesicles. In other embodiments, lipid vesicles comprising one ormore of the cationic lipids described herein are complexed with nucleicacids to form lipoplexes.

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, or from about 70 to about 90 nm. The lipid particles ofthe invention also typically have a lipid:therapeutic agent (e.g.,lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1, fromabout 3:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (SNALP) which comprise aninterfering RNA (e.g., siRNA, Dicer-substrate dsRNA, shRNA, aiRNA,and/or miRNA), a cationic lipid (e.g., one or more cationic lipids ofFormula I-XII or salts thereof as set forth herein), a non-cationiclipid (e.g., mixtures of one or more phospholipids and cholesterol), anda conjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid conjugates). The SNALP may comprise at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified interfering RNAmolecules. Nucleic acid-lipid particles and their method of preparationare described in, e.g., U.S. Pat. Nos. 5,753,613; 5,785,992; 5,705,385;5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication No.WO 96/40964, the disclosures of which are each herein incorporated byreference in their entirety for all purposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a SNALP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the SNALP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the SNALP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

In particular embodiments, the present invention provides a lipidparticle (e.g., SNALP) composition comprising a plurality of lipidparticles described herein and an antioxidant. In certain instances, theantioxidant in the lipid particle composition reduces, prevents, and/orinhibits the degradation of a cationic lipid present in the lipidparticle. In instances wherein the active agent is a therapeutic nucleicacid such as an interfering RNA (e.g., siRNA), the antioxidant in thelipid particle composition reduces, prevents, and/or inhibits thedegradation of the nucleic acid payload, e.g., by reducing, preventing,and/or inhibiting the formation of adducts between the nucleic acid andthe cationic lipid. Non-limiting examples of antioxidants includehydrophilic antioxidants such as chelating agents (e.g., metal chelatorssuch as ethylenediaminetetraacetic acid (EDTA), citrate, and the like),lipophilic antioxidants (e.g., vitamin E isomers, polyphenols, and thelike), salts thereof; and mixtures thereof. If needed, the antioxidantis typically present in an amount sufficient to prevent, inhibit, and/orreduce the degradation of the cationic lipid and/or active agent presentin the particle, e.g., at least about 20 mM EDTA or a salt thereof, orat least about 100 mM citrate or a salt thereof. An antioxidant such asEDTA and/or citrate may be included at any step or at multiple steps inthe lipid particle formation process described in Section VI (e.g.,prior to, during, and/or after lipid particle formation).

Additional embodiments related to methods of preventing the degradationof cationic lipids and/or active agents (e.g., therapeutic nucleicacids) present in lipid particles, compositions comprising lipidparticles stabilized by these methods, methods of making these lipidparticles, and methods of delivering and/or administering these lipidparticles are described in U.S. Provisional Application No. 61/265,671,entitled “SNALP Formulations Containing Antioxidants,” filed Dec. 1,2009, the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

A. Cationic Lipids

Any of the novel cationic lipids of Formula I-XII or salts thereof asset forth herein may be used in the lipid particles of the presentinvention (e.g., SNALP), either alone or in combination with one or moreother cationic lipid species or non-cationic lipid species.

In some embodiments, a mixture of cationic lipids or salts thereof canbe included in the lipid particles of the present invention. In theseembodiments, the mixture of cationic lipids includes a cationic lipid ofFormulas I-XII together with one or more additional cationic lipids.Other cationic lipids suitable for use in combination with the cationiclipids of Formulas I-XII include cationic lipids of Formula XIII havingthe following structure (or salts thereof):

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In some instances, R¹ andR² are both methyl groups. In certain instances, R³ and R⁴ are both thesame, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc. In certain otherinstances, R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C₁₄)and R⁴ is linoleyl (C₁₈). In a preferred embodiment, the cationic lipidof Formula XIII is symmetrical, i.e., R³ and R⁴ are both the same. Inanother preferred embodiment, both R³ and R⁴ comprise at least two sitesof unsaturation. In some embodiments, R³ and R⁴ are independentlyselected from the group consisting of dodecadienyl, tetradecadienyl,hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R³and R⁴ are both linoleyl. In some embodiments, R³ and R⁴ comprise atleast three sites of unsaturation and are independently selected from,e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, andicosatrienyl. In particular embodiments, the cationic lipid of FormulaXIII comprises 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), or mixturesthereof.

In some embodiments, the cationic lipid of Formula XIII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XIII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

Other cationic lipids suitable for use in combination with the cationiclipids of Formulas I-XII include cationic lipids of Formula XIV havingthe following structure (or salts thereof):

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In certain instances, R³and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc.In certain other instances, R³ and R⁴ are different, i.e., R³ istetradectrienyl (C₁₄) and R⁴ is linoleyl (C₁₈). In a preferredembodiment, the cationic lipid of Formula XIV is symmetrical, i.e., R³and R⁴ are both the same. In another preferred embodiment, both R³ andR⁴ comprise at least two sites of unsaturation. In some embodiments, R³and R⁴ are independently selected from the group consisting ofdodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, andicosadienyl. In a preferred embodiment, R³ and R⁴ are both linoleyl. Insome embodiments, R³ and R⁴ comprise at least three sites ofunsaturation and are independently selected from, e.g., dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

In some embodiments, the cationic lipid of Formula XIV forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XIV is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids falling within the scope of Formulas XIII andXIV, is described in U.S. Patent Publication No. 20060083780, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In addition to the cationic lipids of Formulas XIII and XIV, othercationic lipids suitable for use in combination with one or morecationic lipids of Formulas I-XII include, but are not limited to,1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.C1),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-K-DMA; also known asDLin-M-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DODMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide(DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine(DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane(DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and mixtures thereof.

Additional cationic lipids suitable for use in combination with one ormore cationic lipids of Formulas I-XII include, without limitation,cationic lipids such as(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA or “MC3”) and certain analogs thereof asdescribed in U.S. Provisional Patent Application No. 61/334,104,entitled “Novel Cationic Lipids and Methods of Use Thereof,” filed May12, 2010, and PCT Publication Nos. WO 2010/054401, WO 2010/054405, WO2010/054406, and WO 2010/054384, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

The synthesis of cationic lipids such as DO-C-DAP, DMDAP, DOTAP.Cl,DLin-M-K-DMA, as well as additional cationic lipids, is described in PCTPublication No. WO 2010/042877, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA,DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ,DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 09/086,558, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of a number of other cationic lipids and related analogshas been described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96/10390, the disclosures of which are each herein incorporated byreference in their entirety for all purposes. Additionally, a number ofcommercial preparations of cationic lipids can be used, such as, e.g.,LIPOFECTIN® (including DOTMA and DOPE, available from GIBCOIBRL);LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL); andTRANSFECTAM® (including DOGS, available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 45 mol % toabout 90 mol %, from about 45 mol % to about 85 mol %, from about 45 mol% to about 80 mol %, from about 45 mol % to about 75 mol %, from about45 mol % to about 70 mol %, from about 45 mol % to about 65 mol %, fromabout 45 mol % to about 60 mol %, from about 45 mol % to about 55 mol %,from about 50 mol % to about 90 mol %, from about 50 mol % to about 85mol %, from about 50 mol % to about 80 mol %, from about 50 mol % toabout 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol% to about 65 mol %, from about 50 mol % to about 60 mol %, from about55 mol % to about 65 mol % or from about 55 mol % to about 70 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle.

In certain preferred embodiments, the cationic lipid comprises fromabout 50 mol % to about 58 mol %, from about 51 mol % to about 59 mol %,from about 51 mol % to about 58 mol %, from about 51 mol % to about 57mol %, from about 52 mol % to about 58 mol %, from about 52 mol % toabout 57 mol %, from about 52 mol % to about 56 mol %, or from about 53mol % to about 55 mol % (or any fraction thereof or range therein) ofthe total lipid present in the particle. In particular embodiments, thecationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %,54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Incertain other embodiments, the cationic lipid comprises (at least) about66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, or 90 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In additional embodiments, the cationic lipid comprises from about 2 mol% to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10mol % to about 50 mol %, from about 20 mol % to about 50 mol %, fromabout 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %,or about 40 mol % (or any fraction thereof or range therein) of thetotal lipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described in PCTPublication No. WO 09/127,060, U.S. application Ser. No. 12/794,701,filed Jun. 4, 2010, and U.S. application Ser. No. 12/828,189, entitled“Novel Lipid Formulations for Delivery of Therapeutic Agents to SolidTumors,” filed Jun. 30, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by ±5 mol %. For example, in the 1:57 lipid particle (e.g.,SNALP) formulation, the target amount of cationic lipid is 57.1 mol %,but the actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3mol %, ±2 mol %, 1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1mol % of that target amount, with the balance of the formulation beingmade up of other lipid components (adding up to 100 mol % of totallipids present in the particle). Similarly, in the 7:54 lipid particle(e.g., SNALP) formulation, the target amount of cationic lipid is 54.06mol %, but the actual amount of cationic lipid may be ±5 mol %, ±4 mol%, ±3 mol %, ±2 mol %, ±1 mol %, 0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol % of that target amount, with the balance of the formulationbeing made up of other lipid components (adding up to 100 mol % of totallipids present in the particle).

B. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., SNALP) can be any of a variety of neutral uncharged,zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyolphosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127,060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., SNALP) comprises or consists of a mixture of one ormore phospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., SNALP) comprises or consists of one or more phospholipids, e.g.,a cholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., SNALP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol-% to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol (or any fraction thereof) ofthe total, lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. In otherembodiments, the cholesterol component in the mixture comprises about36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereofor range therein) of the total lipid present in the particle. Typically,a 1:57 lipid particle formulation comprising a mixture of phospholipidand cholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Asa non-limiting example, a 1:62 lipid particle formulation may comprisecholesterol at about 37 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:58lipid particle formulation may comprise cholesterol at about 35 mol %(or any fraction thereof) of the total lipid present in the particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described in PCTPublication No. WO 09/127,060, U.S. application Ser. No. 12/794,701,filed Jun. 4, 2010, and U.S. application Ser. No. 12/828,189, entitled“Novel Lipid Formulations for Delivery of Therapeutic Agents to SolidTumors,” filed Jun. 30, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., SNALP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, 0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, ±2 mol %,±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle). Similarly, in the 7:54 lipid particle (e.g., SNALP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.75 mol %, ±0.5mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

C. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., SNALP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the particles comprise either a PEG-lipid conjugateor an ATTA-lipid conjugate together with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-co-methyl-poly(ethyleneglycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S—NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). In otherinstances, the PEG moiety has an average molecular weight of from about550 daltons to about 1000 daltons, from about 250 daltons to about 1000daltons, from about 400 daltons to about 1000 daltons, from about 600daltons to about 900 daltons, from about 700 daltons to about 800daltons, or about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 daltons. In preferred embodiments, thePEG moiety has an average molecular weight of about 2,000 daltons orabout 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (CO, lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula XVIII above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 750 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In other instances, the PEG moiety has an averagemolecular weight of from about 550 daltons to about 1000 daltons, fromabout 250 daltons to about 1000 daltons, from about 400 daltons to about1000 daltons, from about 600 daltons to about 900 daltons, from about700 daltons to about 800 daltons, or about 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 daltons. Inpreferred embodiments, the PEG has an average molecular weight of about2,000 daltons or about 750 daltons. The PEG can be optionallysubstituted with alkyl, alkoxy, acyl, or aryl groups. In certainembodiments, the terminal hydroxyl group is substituted with a methoxyor methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such technique's are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g.,SNALP) of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334; PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

Suitable CPLs include compounds of Formula XIX:A-W—Y  (XIX),wherein A, W, and Y are as described below.

With reference to Formula XIX, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional examples, percentages, and/or ranges of lipid conjugatessuitable for use in the lipid particles of the invention are describedin PCT Publication No. WO 09/127,060, U.S. patent application Ser. No.12/794,701, filed Jun. 4, 2010, U.S. application Ser. No. 12/828,189,entitled “Novel Lipid Formulations for Delivery of Therapeutic Agents toSolid Tumors,” filed Jun. 30, 2010, U.S. Provisional Application No.61/294,828, filed Jan. 13, 2010, U.S. Provisional Application No.61/295, 140, filed Jan. 14, 2010, and PCT Publication No. WO2010/006282, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., SNALP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, ±0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., SNALP)size.

VI. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., SNALP, in which anactive agent or therapeutic agent such as an interfering RNA (e.g.,siRNA) is entrapped within the lipid portion of the particle and isprotected from degradation, can be formed by any method known in the artincluding, but not limited to, a continuous mixing method, a directdilution process, and an in-line dilution process.

In particular embodiments, the cationic lipids may comprise lipids ofFormulas I-XII or salts thereof, alone or in combination with othercationic lipids. In other embodiments, the non-cationic lipids are eggsphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoylphosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via a continuous mixingmethod, e.g., a process that includes providing an aqueous solutioncomprising a nucleic acid (e.g., interfering RNA) in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe nucleic acid within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) produced via a direct dilution process thatincludes forming a lipid vesicle (e.g., liposome) solution andimmediately and directly introducing the lipid vesicle solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of lipid vesiclesolution introduced thereto. As a non-limiting example, a lipid vesiclesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., liposome) solution formed in a first mixing region is immediatelyand directly mixed with dilution buffer in the second mixing region. Inpreferred aspects, the second mixing region includes a T-connectorarranged so that the lipid vesicle solution and the dilution bufferflows meet as opposing 180° flows; however, connectors providingshallower angles can be used, e.g., from about 27° to about 180° (e.g.,about 90°). A pump mechanism delivers a controllable flow of buffer tothe second mixing region. In one aspect, the flow rate of dilutionbuffer provided to the second mixing region is controlled to besubstantially equal to the flow rate of lipid vesicle solutionintroduced thereto from the first mixing region. This embodimentadvantageously allows for more control of the flow of dilution buffermixing with the lipid vesicle solution in the second mixing region, andtherefore also the concentration of lipid vesicle solution in bufferthroughout the second mixing process. Such control of the dilutionbuffer flow rate advantageously allows for small particle size formationat reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., SNALP) can besized by any of the methods available for sizing liposomes. The sizingmay be conducted in order to achieve a desired size range and relativelynarrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., SNALP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., SNALP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making SNALP-CPLs (CPL-containingSNALP) are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during, for example, the SNALPformation steps. The post-insertion technique results in SNALP havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALP having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

VII. Kits

The present invention also provides lipid particles (e.g., SNALP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., SNALP), wherein the particlesare produced by one of the processes set forth herein. In certainembodiments, the kit may further comprise an endosomal membranedestabilizer (e.g., calcium ions). The kit typically contains theparticle compositions of the invention, either as a suspension in apharmaceutically acceptable carrier or in dehydrated form, withinstructions for their rehydration (if lyophilized) and administration.

The lipid particles of the present invention can be tailored topreferentially target particular tissues, organs, or tumors of interest.In some instances, the 1:57 lipid particle (e.g., SNALP) formulation canbe used to preferentially target the liver (e.g., normal liver tissue).In other instances, the 7:54 lipid particle (e.g., SNALP) formulationcan be used to preferentially target solid tumors such as liver tumorsand tumors outside of the liver. In preferred embodiments, the kits ofthe invention comprise these liver-directed and/or tumor-directed lipidparticles, wherein the particles are present in a container as asuspension or in dehydrated form.

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VIII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., SNALP) areuseful for the introduction of active agents or therapeutic agents(e.g., nucleic acids such as interfering RNA) into cells. Accordingly,the present invention also provides methods for introducing an activeagent or therapeutic agent such as a nucleic acid (e.g., interferingRNA) into a cell. In some instances, the cell is a liver cell such as,e.g., a hepatocyte present in liver tissue. In other instances, the cellis a tumor cell such as, e.g., a tumor cell present in a solid tumor.The methods are carried out in vitro or in vivo by first forming theparticles as described above and then contacting the particles with thecells for a period of time sufficient for delivery of the active agentor therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., SNALP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., SNALP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., SNALP)is formed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., SNALP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle may beadministered to the mammal. In some instances, an interfering RNA (e.g.,siRNA) is formulated into a SNALP, and the particles are administered topatients requiring such treatment. In other instances, cells are removedfrom a patient, the interfering RNA is delivered in vitro (e.g., using aSNALP described herein), and the cells are reinjected into the patient.

A. In vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In certainembodiments, the presence of a therapeutic agent such as a nucleic acidis detectable in cells of the lung, liver, tumor, or at a site ofinflammation at about 8, 12, 24, 36, 48, 60, 72 or 96 hours afteradministration. In other embodiments, downregulation of expression of atarget sequence by an interfering RNA (e.g., siRNA) is detectable atabout 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In yetother embodiments, downregulation of expression of a target sequence byan interfering RNA (e.g., siRNA) occurs preferentially in liver cells(e.g., hepatocytes), tumor cells, or in cells at a site of inflammation.In further embodiments, the presence or effect of an interfering RNA(e.g., siRNA) in cells at a site proximal or distal to the site ofadministration or in cells of the lung, liver, or a tumor is detectableat about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16,18, 19, 20, 22, 24, 26, or 28 days after administration. In additionalembodiments, the lipid particles (e.g., SNALP) of the invention areadministered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA) suspended in diluents suchas water, saline, or PEG 400; (b) capsules, sachets, or tablets, eachcontaining a predetermined amount of a therapeutic agent such as nucleicacid (e.g., interfering RNA), as liquids, solids, granules, or gelatin;(c) suspensions in an appropriate liquid; and (d) suitable emulsions.Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA) in aflavor, e.g., sucrose, as well as pastilles comprising the therapeuticagent in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to thetherapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as SNALP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 10⁸-10¹⁰ particles peradministration (e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA) can be to any cell grown inculture, whether of plant or animal origin, vertebrate or invertebrate,and of any tissue or type. In preferred embodiments, the cells areanimal cells, more preferably mammalian cells, and most preferably humancells (e.g., tumor cells or hepatocytes).

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the SNALP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of SNALP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the SNALP or otherlipid particle affects delivery efficiency, thereby optimizing the SNALPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, β-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a SNALP formulationoptimized for an expression plasmid will also be appropriate forencapsulating an interfering RNA. In other instances, an ERP assay canbe adapted to measure downregulation of transcription or translation ofa target sequence in the presence or absence of an interfering RNA(e.g., siRNA). By comparing the ERPs for each of the various SNALP orother lipid particles, one can readily determine the optimized system,e.g., the SNALP or other lipid particle that has the greatest uptake inthe cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, but are not limited to, hepatocytes, hematopoietic precursor(stem) cells, fibroblasts, keratinocytes, endothelial cells, skeletaland smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes,terminally differentiated cells, slow or noncycling primary cells,parenchymal cells, lymphoid cells, epithelial cells, bone cells, and thelike.

In particular embodiments, an active agent or therapeutic agent such asa nucleic acid (e.g., an interfering RNA) is delivered to cancer cells(e.g., cells of a solid tumor) including, but not limited to, livercancer cells, lung cancer cells, colon cancer cells, rectal cancercells, anal cancer cells, bile duct cancer cells, small intestine cancercells, stomach (gastric) cancer cells, esophageal cancer cells,gallbladder cancer cells, pancreatic cancer cells, appendix cancercells, breast cancer cells, ovarian cancer cells, cervical cancer cells,prostate cancer cells, renal cancer cells, cancer cells of the centralnervous system, glioblastoma tumor cells, skin cancer cells, lymphomacells, choriocarcinoma tumor cells, head and neck cancer cells,osteogenic sarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as SNALP encapsulating anucleic acid (e.g., an interfering RNA) is suited for targeting cells ofany cell type. The methods and compositions can be employed with cellsof a wide variety of vertebrates, including mammals, such as, e.g,canines, felines, equines, bovines, ovines, caprins, rodents (e.g.,mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g.monkeys, chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., SNALP) are detectable in the subject at about 8, 12,24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22,24, 25, or 28 days after administration of the particles. The presenceof the particles can be detected in the cells, tissues, or otherbiological samples from the subject. The particles may be detected,e.g., by direct detection of the particles, detection of a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA) sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), or acombination thereof.

1. Detection of Particles

Lipid particles of the invention such as SNALP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic Acids.Res., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

IX. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Synthesis of 1,2-Di-γ-linolenyloxy-N,N-dimethylaminopropane(γ-DLenDMA)

γ-DLenDMA (Compound 1) having the structure shown below was synthesizedas described below.

A 250 mL round bottom flask was charged with3-(dimethylamino)-1,2-propanediol (0.8 g, 6.7 mmol), tetrabutylammoniumhydrogen sulphate (1 g), gamma linolenyl mesylate(cis-6,9,12-octadecatriene sulphonic acid) (5 g, 14.6 mmol), and 30 mLtoluene. After stirring for 15 minutes, the reaction was cooled to 0-5°C. A solution of 40% sodium hydroxide (15 mL) was added slowly. Thereaction was left to stir for approximately 48 hours. An additional 15mL of toluene was then added to the reaction vessel, along with 40%sodium hydroxide (15 mL). After the reaction was stirred for anadditional 12 hours, water (50 mL) and isopropyl acetate (50 mL) wereadded and stirred for 15 minutes. The mixture was then transferred to a500 mL separatory funnel and allowed to separate. The lower aqueousphase was run off and the organic phase was washed with saturated sodiumchloride (2×50 mL). Since the aqueous and organic phases resulting fromthe saturated sodium chloride washes could not be completely separatedafter 20 minutes, the lower aqueous phase (slightly yellow) was run offand back extracted with chloroform (˜45 mL). The organic phase was driedwith MgSO₄, filtered, and the solvent evaporated.

The crude product, an orange liquid, was purified on columnchromatography using silica gel (60 g) with 0-3% methanol gradient indichloromethane to yield 3.19 g. The product was further purified viacolumn chromatography on silica gel (50 g) with 10-30% ethyl acetategradient in hexanes to yield 1.26 g pure product.

Example 2 Synthesis of Asymmetric Cationic Lipids

Asymmetric cationic lipids (Compounds 4-9) having the structures shownbelow were synthesized as shown in the following schematic diagram.

Step 1: Synthesis of 2-Hydroxy-3-linoleyloxypropylbromide (Compound 2)

A 500 ml round bottom flask was charged with epibromohydrin (5.8 g, 42mmol), linoleyl alcohol (15 g, 56 mmol), a stir bar, and then flushedwith nitrogen. Anhydrous DCM (250 ml) was added via cannula, followed byBF₃.Et₂O (0.53 ml, 4.2 mmol), and stirred at 40° C. for 3 hours.Progress of reaction was monitored by TLC to ensure no epoxide wasremaining. The reaction mixture was transferred to a 500 ml separatoryfunnel with DCM (2×50 ml), washed with NaHCO₃ (2×200 ml), water (200ml), brine (200 ml), dried with MgSO₄, and solvent evaporated. The crudeproduct was purified using column chromatography on silica gel (300 g)with 5-15% ether gradient in hexanes to yield 12.6 g.

Step 2: Synthesis of 2-Hydroxy-3-linoleyloxy-N,N-dimethylpropylamine(Compound 3)

A 500 mL round bottom flask was charged with Compound 2(2-Hydroxy-3-linoleyloxypropylbromide) (10 g, 25 mmol) and a stir bar.After flushing with nitrogen, a 2.0 M solution of dimethylamine inmethyl alcohol (250 mL) was added via cannula. The resulting mixture wasstirred at room temperature for 48 hours. The progress of the reactionwas monitored using TLC. The reaction solution was evaporated and thecrude product (9.1 g) used without further purification.

Synthesis of Linoleyl/Oleyl DMA (Compound 4)

A 250 mL round bottom flask was charged with2-Hydroxy-3-linoleyloxy-N,N-dimethylpropylamine (1.5 g, 4.1 mmol), oleylmethane sulfonate (2.14 g, 6.2 mmol), toluene (30 mL), and a stir bar.The resulting mixture was cooled to 0-5° C. While stirring, a solutionof 40% NaOH (15 mL) was added, followed by 1.0 M solution of tertbutylammonium hydroxide ((TBAH) 1.5 ml, 1.5 mmol). The reaction was stirredat room temperature for 60 hours and progress of reaction was monitoredusing TLC. Water (50 ml) and isopropyl acetate (50 ml) were added andthe resulting solution stirred for 10 minutes. The mixture wasquantitatively transferred to a 500 ml separatory funnel with isopropylacetate (10 ml), and the lower aqueous phase was run off. The organicphase was washed with water (2×50 ml), dried with MgSO₄, filtered, andsolvent removed. During the second wash, ethanol (25 ml) was added toensure complete separation of the two phases. The aqueous phases werepooled and back extracted with chloroform. The crude product (4.1 g) waspurified by column chromatography on silica gel (60 g) with 0-3%methanol gradient in dichloromethane to yield 0.45 g.

Synthesis of Linoleyl/Phytanyl DMA (Compound 5)

Compound 5 was synthesized analogously to Compound 4, but substitutingoleyl mesylate for phytanyl methane sulfonate (2.26 g, 6.2 mmol) in thealkylation step. Final yield obtained: 0.46 g.

Synthesis of Linoleyl/Linolenyl DMA (Compound 6)

Compound 6 was synthesized analogously to Compound 4, but substitutingoleyl mesylate for linolenyl methane sulfonate (2.12 g, 6.2 mmol) in thealkylation step. Final yield obtained: 0.49 g.

Synthesis of Linoleyl/Stearyl DMA (Compound 7)

Compound 7 was synthesized analogously to Compound 4, but substitutingoleyl mesylate for stearyl methane sulfonate (2.16 g, 6.2 mmol) in thealkylation step. Final yield obtained: 0.77 g.

Synthesis of Linoleyl/C₆:0 DMA (Compound 8)

Compound 8 was synthesized analogously to Compound 4, but substitutingoleyl mesylate for hexanyl methane sulfonate (1.12 g, 6.2 mmol) in thealkylation step. Final yield obtained: 0.18 g.

Synthesis of Linoleyl/C₆:1 DMA (Compound 9)

A 100 mL round bottom flask was charged with a stir bar, NaH (0.4 g, 17mmol), and 35 mL benzene. Subsequently,2-Hydroxy-3-linoleyloxy-N,N-dimethylpropylamine (1.20 g, 3.3 mmol) wasadded followed immediately by cis-3-hexenyl methane sulfonate (0.76 g,4.3 mmol). The reaction was flushed with nitrogen and stirred overnightat 50° C. Progress of the reaction was monitored via TLC. The reactionmixture was transferred to a 250 mL separatory funnel and diluted withbenzene to a final volume of 100 mL. The reaction was quenched withethanol (60 mL) and then washed with water (100 mL). The lower aqueousphase was run off and the reaction mixture washed again with ethanol (60mL) and water (100 mL). The organic phase was dried with MgSO₄,filtered, and solvent removed. The crude product (0.83 g) was purifiedby column chromatography on silica gel (30 g) with 0-3% methanolgradient in dichloromethane to yield 0.17 g.

Example 3 Synthesis of Cationic Lipids of the TLinDMA Family

The following diagram provides a general scheme for synthesizing membersof the C(n)-TLinDMA family of cationic lipids:

TLinDMA(1-(2,3-linoleyloxypropoxy)-2-(linoleyloxy)-(N,N-dimethyl)-propyl-3-amine)(Compound 12) was synthesized as follows:

Synthesis of Compound 10

A 1000 ml round bottom flask was charged with epibromohydrin (5 g, 37mmol), glycerol (10 g, 110 mmol), a stir bar and then flushed withnitrogen. Anhydrous chloroform (350 mL) was added via cannula, followedby BF₃.Et₂O (0.5 mL, 3.7 mmol) and refluxed for 3 hours under nitrogen.The reaction mixture was cooled and subsequently stirred at roomtemperature overnight. Upon completion of the reaction, the reactionmixture was concentrated and the crude product (15 g) was purified viacolumn chromatography using silica gel (150 g).

Synthesis of Compound 11

A 500 mL round bottom flask was charged with Compound 10 (3.8 g, 17mmol) and a stir bar. After flushing with nitrogen, dimethylamine in a2.0 M methyl alcohol solution (170 mL) was added via cannula. Theresulting mixture was stirred at room temperature for 48 hours. Theprogress of the reaction was monitored using TLC. The crude product wasused without further purification.

Synthesis of TLinDMA (Compound 12)

A 100 mL round bottom flask was charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Subsequently, Compound 11 (0.4 g, 2 mmol) wasadded followed immediately by linoleyl methane sulfonate (2.8 g, 8mmol). The reaction was flushed with nitrogen and refluxed overnight.Progress of the reaction was monitored via TLC. The reaction mixture wastransferred to a 250 mL separatory funnel and diluted with benzene to afinal volume of 50 mL. The reaction was quenched with ethanol (30 mL)and then washed with water (50 mL). The lower aqueous phase was run offand the reaction mixture was washed again with ethanol (30 mL) and water(50 mL). The organic phase was dried with MgSO₄, filtered, and solventremoved. The crude product (2.3 g) was purified via columnchromatography on silica gel (60 g) with 0-3% methanol gradient indichloromethane.

C2-TLinDMA (Compound 16) was synthesized as follows Synthesis ofCompound 13

A solution of 4-bromo-1-butene (11.5 g, 85 mmol) in CH₂Cl₂ (anh., 120ml) was prepared under nitrogen in a 1000 ml RBF with a magneticstirrer. In a separate flask, a solution of 3-chloroperbenzoic acid(77%, MW 173, 44.05 g, 196 mmol) in CH₂Cl₂ (anh., 250 ml) prepared andadded to the reaction mixture by canulla. The reaction was stirred for 3days, and then concentrated. The product (oil/white solid mixture) wasre-dissolved in THF (300 mL) and a solution of 4% sodium dithionite (180mL) added to remove excess peracid. The mixture (now cloudy) was stirredfor 20 minutes and then EtOAc (750 mL) added. The mixture wastransferred to a separating funnel and the organic was washed with water(100 mL), sat. NaHCO₃ (2×300 mL, EFFERVESCENCE), water again (300 mL)and brine (300 mL). The solution was concentrated and the productpurified by chromatography.

Synthesis of Compound 14

A 250 ml round bottom flask was charged with Compound 13 (1.3 g, 9mmol), glycerol (2.5 g, 27 mmol), a stir bar and then flushed withnitrogen. Anhydrous chloroform (100 mL) was added via cannula, followedby BF₃.Et₂O (0.15 mL, 1.1 mmol) and refluxed for 3 hours under nitrogen.The reaction mixture was subsequently stirred at room temperatureovernight.

Synthesis of Compound 15

A 50 mL round bottom flask was charged with Compound 14 (0.3 g, 1.2mmol) and a stir bar. After flushing with nitrogen, dimethylamine in a2.0 M methyl alcohol solution (25 mL) was added via syringe. Theresulting mixture was stirred at room temperature for 48 hours. Theprogress of the reaction was monitored using t.l.c. The reaction mixturewas concentrated and the crude product used without furtherpurification.

Synthesis of C2-TLinDMA (Compound 16)

A 100 mL round bottom flask was charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Compound 15 (0.37 g, 1.8 mmol) was addedfollowed immediately by linoleyl methane sulfonate (2.8 g, 8 mmol). Thereaction was refluxed overnight and progress of the reaction wasmonitored via t.l.c. The reaction mixture was transferred to a 250 mLseparatory funnel and diluted with benzene to a final volume of 50 mL.The reaction was quenched with ethanol (30 mL) and then washed withwater (50 mL). The lower aqueous phase was run off and the reactionmixture washed again with ethanol (30 mL) and water (50 mL). The organicphase was dried with MgSO₄, filtered, and solvent removed. The crudeproduct, 2.5 g, was purified using column chromatography on silica gel(60 g), eluted with 0-3% methanol gradient in DCM.

C3-TLinDMA (Compound 20) was synthesized as follows:

Synthesis of Compound 17

A solution of 5-bromo-1-pentene (85 mmol) in CH₂Cl₂ (anh., 120 ml) isprepared under nitrogen in a 1000 ml RBF with a magnetic stirrer. In aseparate flask, a solution of 3-chloroperbenzoic acid (77%, MW 173,44.05 g, 196 mmol) in CH₂Cl₂ (anh., 250 ml) is prepared and added to thereaction mixture by canulla. The reaction is stirred for 3 days, andthen concentrated. The product (oil/white solid mixture) is re-dissolvedin THF (300 mL) and a solution of 4% sodium dithionite (180 mL) added toremove excess peracid. The mixture (now cloudy) is stirred for 20minutes and then EtOAc (750 mL) added. The mixture is transferred to aseparating funnel and the organic is washed with water (100 mL), sat.NaHCO₃ (2×300 mL, EFFERVESCENCE), water again (300 mL) and brine (300mL). The solution is concentrated and the product purified bychromatography.

Synthesis of Compound 18

A 250 ml round bottom flask is charged with Compound 17 (1.3 g, 9 mmol),glycerol (2.5 g, 27 mmol), a stir bar and then flushed with nitrogen.Anhydrous chloroform (100 mL) is added via cannula, followed by BF₃.Et₂O(0.15 mL, 1.1 mmol) and refluxed for 3 hours under nitrogen. Thereaction mixture is subsequently stirred at room temperature overnight.

Synthesis of Compound 19

A 50 mL round bottom flask is charged with Compound 18 (0.3 g, 1.2 mmol)and a stir bar. After flushing with nitrogen, dimethylamine in a 2.0 Mmethyl alcohol solution (25 mL) is added via syringe. The resultingmixture is stirred at room temperature for 48 hours. The progress of thereaction is monitored using t.l.c. The reaction mixture is concentratedand the crude product used without further purification.

Synthesis of Compound 20

A 100 mL round bottom flask is charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Compound 19 (0.37 g, 1.8 mmol) is addedfollowed immediately by linoleyl methane sulfonate (2.8 g, 8 mmol). Thereaction is refluxed overnight and progress of the reaction monitoredvia t.l.c. The reaction mixture is transferred to a 250 mL separatoryfunnel and diluted with benzene to a final volume of 50 mL. The reactionis quenched with ethanol (30 mL) and then washed with water (50 mL). Thelower aqueous phase is run off and the reaction mixture washed againwith ethanol (30 mL) and water (50 mL). The organic phase is dried withMgSO₄, filtered, and solvent removed. The crude product, 2.5 g, ispurified using column chromatography on silica gel (60 g), eluted with0-3% methanol gradient in DCM.

Example 4 Synthesis of1,2-Diarachidonyloxy-(N,N-dimethyl)-propyl-3-amine (DAraDMA)

DAraDMA (Compound 21) having the structure shown below was synthesizedas described below.

A 250 mL round bottom flask was charged with3-(dimethylamino)-1,2-propanediol (0.3 g, 2.5 mmol), tetrabutylammoniumhydrogen sulphate (0.4 g), arachidonyl methane sulfonate (2 g, 5.4mmol), and 30 mL toluene. After stirring for 15 minutes, the reactionwas cooled to 0-5° C. A solution of 40% sodium hydroxide (15 mL) wasadded slowly. The reaction was left to stir for approximately 48 hours.After 48 hours of stirring, water (50 mL) and isopropyl acetate (50 mL)were added and stirred for 15 minutes. The mixture was then transferredto a 500 mL separatory funnel, quantitatively transferred with (2×5 mLtoluene) and allowed to separate. The lower aqueous phase was run offand the organic phase was washed with saturated sodium chloride (50 mL).Approximately 20 minutes were allowed for phase separation. The twoaqueous phases were separately and consecutively back extracted withchloroform (50 mL). The organic phase was subsequently dried with MgSO₄,filtered, and the solvent evaporated. The crude product (2.6 g) waspurified on column chromatography using silica gel (60 g) with 0-2%methanol gradient in dichloromethane.

Example 5 Synthesis of 1,2-Diphytanyloxy-3-(N,N-dimethyl)-propylamine(DPanDMA)

DPanDMA (Compound 24) having the structure shown below was synthesizedas described below.

Step 1: Synthesis of Phytanol (Compound 22)

Phytol (21.0 g, 70.8 mmol), ethanol (180 mL) and a stir bar were addedto a 500 mL round bottom flask. Raney Nickel 2800 (as purchased, a 50%by weight solution in water if used as purchased, Nickel >89% metalpresent) (6.8 g, 51.5 mmol) was added, and the flask sealed and flushedwith hydrogen. A 12″ needle was used to bubble hydrogen through thesolution for 10 minutes. The reaction was stirred for 5 days, using aballoon as a hydrogen reservoir. Hydrogen was also bubbled through thereaction mixture at 24 h and 48 h, 5 minutes each time. The metalcatalyst was then removed by filtering through Celite. The ethanolicsolution was concentrated, and 200 mL of DCM added to the resulting oil.The solution was washed with water (2×100 mL), dried over MgSO₄, andconcentrated. TLC indicated formation of the phytanol product, yield20.0 g.

Step 2: Synthesis of Phytanyl Mesylate (Compound 23)

Phytanol (22) (20.0 g, 66.7 mmol), triethylamine (18.6 mL, 133 mmol),and a stir bar were added to a 1000 mL round bottom flask. The flask wassealed and flushed with nitrogen. Anhydrous DCM (250 mL) was added, andthe mixture cooled to −15° C. (ice and NaCl). Mesyl Chloride (10.4 mL,133 mmol) was added slowly via syringe over a 30 minute period, and thereaction stirred at −15° C. for a further 1.5 hours. At this point TLCshowed that the starting material had been used up. The solution wasdiluted with DCM (250 mL) and washed with saturated NaHCO₃ (2×200 mL).The organic phase was then dried (MgSO₄), filtered, and concentrated(rotovap). The crude product was purified by column chromatography.Yield: 21.5 g, 85.7%.

Synthesis of DPanDMA (Compound 24)

Sodium hydride (2.5 g, 100 mmol) was added to a 250 mL round bottomflask, along with benzene (40 mL) and a stir bar. In a 50 mL beaker, asolution was made from the N,N-Dimethyl-3-aminopropane-1,2-diol (1.42 g,12 mmol) and benzene (60 mL). This was added to the reaction vessel andthe reaction stirred for 10 minutes (effervescence). Phytanyl Mesylate(23) (10.52 g, 28 mmol) was added and the flask fitted with a condenser,flushed with nitrogen, and heated to reflux. After 18 hours, the flaskwas removed from the heat and allowed to cool. The volume was made up to200 mL with benzene. EtOH was added slowly to quench unreacted sodiumhydride. Once quenching was complete, the reaction mixture was washedtwice with EtOH/H₂O, in a ratio to the benzene of 1:1:0.6benzene:water:ethanol. The aqueous phases were combined and extractedwith CHCl₃ (2×100 mL). Finally, the organic phase was dried (MgSO₄),filtered, and concentrated (rotovap). Purification by columnchromatography yielded DPanDMA as a pale yellow oil (6.1 g, 8.97 mmol,74.7%).

Example 6 Synthesis of Novel Lipids with Alternate Head Groups

Novel lipids with alternate head groups (Compounds 28-33) having thestructures shown below were synthesized as shown in the followingschematic diagram.

Step 1: Synthesis of 1,2-Dilinoleyloxy-3-allyloxypropane (Compound 25)

In a 2000 mL round bottom flask equipped with a large magnetic stir bar,3-allyloxypropane-1,2-diol (5.94 g, 45 mmol), and tetrabutylammoniumhydrogen sulfate (6 g, 18 mmol) were added. A solution of linoleylmesylate (33.8 g, 98 mmol) in toluene (150 mL) was also made and added.The mixture was cooled to 0-5° C. with an ice bath. 50% sodium hydroxide(70 mL) was then slowly added, rinsing in with deionized water (20 mL).The resulting mixture is stirred at room temperature, under nitrogen for40 hours. Deionized water (200 mL) and isopropyl acetate (200 mL) wereadded and the mixture stirred vigorously for a further 10-15 min. Themixture was transferred to a 1000-mL separating funnel and allowed toseparate. The aqueous phase was removed and the organic phase washedwith saturated sodium chloride solution (2×150 mL). The organic phasewas dried with magnesium sulfate (40 g), filtered, and concentrated toobtain a neat orange oil. The oil was purified by column chromatography.Final yield: 19.5 g (69%).

Step 2: Synthesis of 1,2-Dilinoleylglycerol (Compound 26)

1,2-Dilinoleyloxy-3-allyloxypropane (25) (18 g, 28.6 mmol) was dissolvedin ethanol (150 mL) in a 500 mL round bottom flask with a stir bar.Trifluoroacetic acid (15 mL) and tetrakis (triphenylphosphine)palladium(0) (5.0 g, 4.3 mmol) were added and the flask flushed withnitrogen. A water condenser was fitted and the flask wrapped tin foil toreduce exposure to light. The reaction was refluxed under nitrogenovernight. The solution was concentrated to an orange oil, thenredissolved in DCM (250 mL), and washed with water (2×200 mL) andsaturated brine (1×200 mL). The aqueous phases were combined andextracted with DCM (2×100 mL). Organic phases were combined, dried overMgSO₄ and concentrated. The crude product was purified by columnchromatography to yield 1,2-dilinoleylglycerol as a colorless oil (11.0g, 18.7 mmol, 65.3%).

Step 3: Synthesis of 1,2-Dilinoleyl-3-methanesulfonylglycerol (Compound27)

1,2-Dilinoleylglycerol (26) (11.0 g, 18.7 mmol), triethylamine (5.04 mL,36 mmol), and a stir bar were added to a 250 mL round bottom flask. Theflask was sealed and flushed with nitrogen. Anhydrous DCM (75 mL) wasadded, and the mixture cooled to −15° C. (Ice and NaCl). Mesyl Chloride(2.80 mL, 36 mmol) was added via syringe over a 5 minute period, and thereaction stirred at −15° C. for 1 hour. At this point TLC showed thatthe starting material had been consumed. The solution was diluted withDCM (120 mL) and washed with saturated NaHCO₃ (2×150 mL). The organicphase was then dried (MgSO₄), filtered, concentrated, and the productpurified by column chromatography (10.6 g, 85%).

Synthesis of 1,2 Dilinoleyloxy 3-piperidinopropylamine (DLinPip)(Compound 28)

1,2-Dilinoleyl-3-methanesulfonylglycerol (27) (1.0 g, 1.5 mmol) and astir bar were added to a 50 mL RBF and flushed with nitrogen. Ethanol(15 mL) and piperidine (1.28 g, 1.48 mL, 15 mmol) were added and thereaction stirred for 48 h at 50° C. The reaction mixture wasconcentrated (by rotovap) to a volume of 5-10 mL, then ethyl acetate (50mL) added (a precipitate formed). The mixture was washed with NaHCO₃(2×50 mL), and water (2×50 mL). The precipitate was washed out. Theaqueous phase was extracted once with DCM (30 mL), then the organicscombined, dried (MgSO₄), and concentrated to yield a yellow oil, whichwas purified by column chromatography (500 mg, 51%).

Synthesis of 1,2 Dilinoleyloxy 3-(3′-hydroxypiperidino)-propylamine(DLinPip-30H) (Compound 29)

1,2-Dilinoleyl-3-methanesulfonylglycerol (27) (1.0 g, 1.5 mmol) and astir bar were added to a 50 mL RBF and flushed with nitrogen. Ethanol(15 mL) and 3-hydroxypiperidine (1.52 g, 15 mmol, 10-fold excess) wereadded and the reaction stirred for 48 h at 50° C. The reaction mixturewas concentrated (by rotovap), then ethyl acetate (30 mL) added (aprecipitate formed). The mixture was washed with NaHCO₃ (2×30 mL), andwater (2×30 mL) and brine (30 mL). The precipitate was washed out. Theorganic was dried (MgSO₄) and concentrated to yield a yellow oil, whichwas purified by column chromatography (450 mg, 45%).

Synthesis of 1,2 Dilinoleyloxy 3-(4′-hydroxypiperidino)-propylamine(DLinPip-40H) (Compound 30)

1,2-Dilinoleyl-3-methanesulfonylglycerol (27) (1.0 g, 1.5 mmol) and astir bar were added to a 50 mL RBF and flushed with nitrogen. Ethanol(15 mL) and 4-hydroxypiperidine (1.52 g, 15 mmol, 10-fold excess) wereadded and the reaction stirred for 48 h at 50° C. The reaction mixturewas concentrated (by rotovap), then ethyl acetate (30 mL) added (aprecipitate formed). The mixture was washed with NaHCO₃ (2×30 mL), andwater (2×30 mL) and brine (30 mL). The precipitate was washed out. Theorganic was dried (MgSO₄) and concentrated to yield a yellow oil, whichwas purified by column chromatography (470 mg, 47%).

Synthesis of 1,2 Dilinoleyloxy 3-(N,N dimethyl)-propylamine (DLinDEA)(Compound 31)

1,2-Dilinoleyl-3-methanesulfonylglycerol (27) (1.0 g, 1.5 mmol) and astir bar were added to a 50 mL RBF and flushed with nitrogen. Ethanol(15 mL) and diethylamine (1.55 mL, 15 mmol) were added and the reactionstirred for 48 h at 40° C. The reaction mixture was concentrated (byrotovap), then ethyl acetate (30 mL) added (a precipitate formed). Themixture was washed with NaHCO₃ (2×30 mL), and water (2×30 mL) and brine(30 mL) The precipitate was washed out. The organic was dried (MgSO₄)and concentrated to yield a yellow oil, which was purified by columnchromatography (590 mg, 61%).

Synthesis ofN1-(2,3-linoleyloxy)propyl)-N1,N3,N3-trimethylpropane-1,3-diamine(2N-DLinDMA) (Compound 32)

1,2-Dilinoleyl-3-methanesulfonylglycerol (27) (1.50 g, 2.25 mmol) and astir bar were put in a 100 mL round-bottom flask. A solution ofN,N,N′-trimethyl-1,3-propanediamine (2.75 g, 23.7 mmol) in methanol (30mL) was added, then the mixture heated to reflux for 48 hours. Thereaction mixture was diluted with CHCl₃ (50 mL), and washed with 1M NaOH(50 mL). Separation was aided by further addition of MeOH. The reactionwas washed 2 more times with dH₂O (25 mL). The aqueous phases wereextracted with CHCl₃ (20 mL), and all organics combined. The organic wasdried over MgSO₄, filtered, concentrated, and purified by columnchromatography to yield a yellow oil (610 mg, 39%).

Example 7 Synthesis of1,2-Didocosahexaenyloxy-(N,N-dimethyl)-propyl-3-amine (DDocDMA)

DDocDMA (Compound 33) having the structure shown below was synthesizedas described below.

A 250 mL round bottom flask was charged with3-(dimethylamino)-1,2-propanediol (0.28 g, 2.36 mmol),tetrabutylammonium hydrogen sulphate (0.4 g), docosahexaenoyl methanesulfate (2.0 g, 5.1 mmol), and 30 mL toluene. After stirring for 15minutes, the reaction was cooled to 0-5° C. A solution of 40% sodiumhydroxide (15 mL) was added slowly. The reaction was left to stir forapproximately 40 hours. After 40 hours of stirring, water (50 mL) andisopropyl acetate (50 mL) were added and stirred for 15 minutes. Themixture was then transferred to a 500 mL separatory funnel,quantitatively transferred with (2×5 mL toluene) and allowed toseparate. The lower aqueous phase was run off and the organic phase waswashed with saturated sodium chloride (50 mL). Approximately 20 minuteswere allowed for phase separation. The two aqueous phases wereseparately and consecutively back extracted with chloroform (50 mL). Theorganic phase was subsequently dried with MgSO₄, filtered, and thesolvent evaporated. The crude product (4.8 g) was purified on columnchromatography using silica gel (60 g) with 0-2% methanol gradient indichloromethane.

Example 8 Synthesis of Novel C2 Lipids

Novel C2 lipids (Compounds 38-40) having the structures shown below weresynthesized as shown in the following schematic diagram.

Step 1: Synthesis of4-(2-Methanesulfonylethyl)-2,2-dimethyl-1,3-dioxolane (34)

4-(2-Hydroxylethyl)-2,2-dimethyl-1,3-dioxolane (25 g, 170 mmol),triethylamine (55.9 mL, 400 mmol), and a stir bar were added to a 1000mL round bottom flask. The flask was sealed and flushed with nitrogen.Anhydrous DCM (600 mL) was added, and the mixture cooled to approx −5°C. (ice and NaCl). Mesyl chloride (19.9 mL, 255 mmol, 1.5 eq) was addedslowly via syringe over a 60 minute period, and the reaction stirred at−5° C. for a further 1.5 hours. At this point TLC showed that thestarting material had been consumed. The solution was diluted with DCM(350 mL), divided into two (˜500 mL) portions, and each portion workedup as follows: the solution was transferred to a 1000-mL separatingfunnel and washed with saturated NaHCO₃ (2×200 mL). The organic phasewas then dried (MgSO₄), filtered, and concentrated (rotovap). The crudeproduct was purified by column chromatography. Final yield: 32.0 g,84.1%.

Step 2: Synthesis of 4-(2-Bromoethyl)-2,2-dimethyl-1,3-dioxolane(Compound 35)

Magnesium bromide etherate (40 g, 130 mmol) and a stir bar were added toa 2000 mL round bottom flask and flushed with nitrogen. A solution of4-(2-methanesulfonylethyl)-2,2-dimethyl-1,3-dioxolane (34) (17.5 g, 78mmol) in anhydrous diethyl ether (900 mL) was added via canulla, and thesuspension stirred overnight. The ether was first decanted into abeaker. Water (200 mL) and ether (300 mL) were added to the precipitateand stirred for 5 minutes. The precipitate was dissolved, and the etherphase was then collected and added to the ether solution from thereaction. The organic phase was then washed, concentrated to about 500mL, washed with water, dried over anhydrous Mg₂SO₄, filtered, andconcentrated to yield a yellow oil (16.0 g). This was purified by flashchromatography to yield 10.6 g of product (50.7 mmol, 65%).

Step 3: Synthesis of 4-Bromobutane-1,2-diol (Compound 36)

4-(2-Bromoethyl)-2,2-dimethyl-1,3-dioxolane (35) (9 g, 43 mmol) wasadded to a 500 mL RBF with a stirbar. 100 mL of MeOH:H₂O:HCl in a ratioof (60:20:5) were added. After 30 minutes, sat. NaHCO₃ (˜75 mL) wasadded (effervescence), until pH paper indicated solution was basic. Atthis point the mixture was slightly cloudy. Ether (300 mL) was added(while stirring) and the cloudiness disappeared. The reaction mixturewas transferred to a 1000 mL sep funnel and the 2 phases separated. Theextraction of the aqueous phase was repeated two more times (2×300 mLether). Organics were combined, dried over MgSO₄ and concentrated toyield a colorless oil (7.0 g), which was purified by columnchromatography.

Step 4: Synthesis of 4-(Dimethylamino)-1,2-butanediol (Compound 37)

4-Bromobutane-1,2-diol (36) (1 g, 6.0 mmol) was added to a 50 mL RBFwith a stir bar, sealed, and flushed with nitrogen. 30 mL ofDimethylamine (2.0M solution in MeOH) was delivered by canulla and thereaction stirred overnight. TLC indicated all the starting material haddisappeared. The solvent (and DMA) were removed by evaporation and thecrude product used without further purification.

Synthesis of 1,2-Dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA)(38)

4-(Dimethylamino)-1,2-butanediol (37) (1.3 g, 3.4 mmol), linoleylmesylate (2.0 g, 5.8 mmol), tetrabutylammonium hydrogen sulphate (0.5 g,1.5 mmol), toluene (30 mL), and a stir bar were added to a 100 mL RBF.30 mL of 40% NaOH was made and added to the reaction mixture. Theresulting mixture was stirred at room temperature, under nitrogen for 60hours. Deionized water (50 mL) and isopropyl acetate (50 mL) were addedand the mixture stirred vigorously for a further 10-15 min. The mixturewas transferred to a 250 mL separating funnel and allowed to separateand the aqueous phase removed. The organic phase was washed twice withwater (2×30 mL) using MeOH to aid the separation, and the organic phasewas dried (MgSO₄), filtered, and concentrated to obtain a dark yellowoil. The oil was purified by column chromatography.

Synthesis of 1,2-Diphytanyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DPanDMA)(39)

Sodium hydride (360 mg, 15 mmol), benzene (40 mL), and a stir bar wereadded to a 50 mL round bottom flask. 4-(Dimethylamino)-1,2-butanediol(37) (200 mg, 1.5 mmol) was added and the reaction stirred for 10minutes (effervescence). Phytanyl Mesylate (23), (1.07 g, 2.92 mmol) wasthen added and the flask fitted with a condenser, flushed with nitrogen,and heated to reflux. After 18 hours, the flask was allowed to cool toroom temperature. The volume was made up to 40 mL with benzene. EtOH wasadded slowly to quench unreacted sodium hydride. Once quenching wascomplete, the reaction mixture was washed twice with an EtOH/H₂O, in aratio to the benzene of 1:1:0.6 benzene:water:ethanol. The aqueouswashes were combined and extracted with CHCl₃ (2×20 mL). Finally, theorganics were combined, dried (MgSO₄), filtered, and concentrated(rotovap). Purification by column chromatography yielded a pale yellowoil (250 mg, 0.145 mmol, 25%).

Synthesis of 1,2-Dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine(C2-DLinDAP) (40)

A flask containing 4-(Dimethylamino)-1,2-butanediol (37) (crude, 266 mg,2 mmol (max)), TEA (0.84 mL, 6 mmol), and DMAP (24 mg, 0.2 mmol) wasflushed with nitrogen before the addition of anhydrous CH₂Cl₂ (50 ml).Linoleoyl chloride (1.2 g, 4 mmol) was added and the solution stirredovernight. The solution was rinsed into a 250 mL separatory funnel withDCM 70 mL) and washed with water (2×50 mL). The organic was dried(MgSO₄), concentrated, and purified by chromatography.

Example 9 Synthesis of Novel Phytanyl Cationic Lipids

DPan-C2K-DMA (Compound 47), DPan-C1K6-DMA (Compound 50), andDPan-C3K-DMA (Compound 54) having the structures shown below weresynthesized as shown in the following schematic diagram.

Synthesis of Phytanol (Compound 22)

Phytol (21.0 g, 70.8 mmol), ethanol (180 mL) and a stir bar were addedto a 500 mL round bottom flask. Raney Nickel 2800 (as purchased, a 50%by weight solution in water if used as purchased, Nickel >89% metalpresent) (6.8 g, 51.5 mmol) was added, and the flask sealed and flushedwith hydrogen. A 12″ needle was used to bubble hydrogen through thesolution for 10 minutes. The reaction was stirred for 5 days, using aballoon as a hydrogen reservoir. Hydrogen was also bubbled through thereaction mixture at 24 h and 48 h, 5 minutes each time. The metalcatalyst was then removed by filtering through Celite. The ethanolicsolution was concentrated, and 200 mL of DCM added to the resulting oil.The solution was washed with water (2×100 mL), dried over MgSO₄, andconcentrated. TLC indicated formation of the phytanol product, yield20.0 g.

Synthesis of Phytanyl Mesylate (Compound 23)

Phytanol (22) (20.0 g, 66.7 mmol), triethylamine (18.6 mL, 133 mmol) anda stir bar were added to a 1000 mL round bottom flask. The flask wassealed and flushed with nitrogen. Anhydrous DCM (250 mL) was added, andthe mixture cooled to −15° C. (Ice and NaCl). Mesyl Chloride (10.4 mL,133 mmol) was added slowly via syringe over a 30 minute period, and thereaction stirred at −15° C. for a further 1.5 hours. At this point TLCshowed that the starting material had been used up. The solution wasdiluted with DCM (250 mL) and washed with saturated NaHCO₃ (2×200 mL).The organic phase was then dried (MgSO₄), filtered and concentrated(rotovap). The crude product was purified by column chromatography.Yield 21.5 g, 85.7%.

Synthesis of Phytanyl Bromide (Compound 41)

Magnesium bromide etherate (17 g, 55 mmol) and a stir bar were added toa 500 mL round bottom flask. The flask was sealed and flushed withnitrogen and anhydrous diethyl ether (200 mL) added via cannula. Asolution of phytanyl mesylate (23) (10.9 g, 28.9 mmol (FW=377)) inanhydrous ether (50 mL) was also added via canulla, and the suspensionstirred overnight. The following morning a precipitate had formed on theside of the flask. Chilled water (200 mL) was added (ppte dissolved) andthe mixture transferred to a 1000-mL separating funnel. After shaking,the organic phase was separated. The aqueous phase was then extractedwith ether (2×150 mL) and all ether phases combined. The ether phase waswashed with water (2×150 mL), brine (150 mL) and dried over anhydrousMg₂SO₄. The solution was filtered, concentrated, and purified by flashchromatography. Final yield 9.5 g (26.3 mmol, 91.1%).

Synthesis of Compound 42

Magnesium turnings (720 mg, 30 mmol), a crystal of iodine, and a stirbarwere added to a 500 mL round-bottom flask. The flask was flushed withnitrogen and anhydrous diethyl ether (200 mL) added via cannula. Asolution of phytanyl bromide (41) (9.5 g, 26.3 mmol) in anhydrous ether(20 mL) was added and the resulting cloudy mixture refluxed overnight.The mixture was cooled to RT and, without removing the subaseal orcondenser, ethyl formate (2.2 g, 2.41 mL, 30 mmol) added via syringe and12″ needle. The addition was made dropwise, directly into the reactionmixture, and the cloudy suspension again stirred overnight. R.M. wastransferred to a 500-mL sep. funnel with ether (50 mL), and washed with10% H₂SO₄ (100 mL—the cloudy R.M. now clarified upon shaking), water(2×100 mL) and brine. The organic was dried over anhydrous Mg₂SO₄,filtered, and concentrated. Yield (crude) was 8 g. TLC indicated thatthe majority of product was the diphytanylmethyl formate, which waspurified by chromatography (0-6% ether in hexane).

Synthesis of Compound 43

The purified formate (42) (5.5 g, 8.86 mmol) was then transferred to a1000 mL round bottom flask with stirbar and 90% EtOH (500 mL) and KOH(2.0 g, 35.7 mmol) added. The reaction mixture was clear, and wasstirred overnight. The following day the mixture was concentrated byrotovap to 50% of its volume and then poured into 200 mL of 5% HCl. Theaqueous phase was extracted with ether (3×100 mL). The combined etherextracts were washed with water (3×200 mL), dried (MgSO₄), andconcentrated. TLC (DCM) revealed reaction to have gone cleanly tocompletion, and the product (5.5 g, 100%) was used without furtherpurification.

Synthesis of Compound 44

To a mixture of Compound 43 (5.5 g, 9.3 mmol), pyridinium chlorochromate(PCC) (5.5 g, 25.5 mmol) and anhydrous sodium carbonate (0.6 g, 5.66mmol) in DCM were added. The resulting suspension was stirred for 1 h,but TLC indicated still some starting material (SM) remaining. Thesuspension was stirred another hour, and appeared to have progressedslightly, but not to completion. Further PCC (1.0 g) and sodiumcarbonate (0.2 g) were added and the reaction stirred overnight.Reaction had now gone to completion. Ether (300 mL) was then added tothe mixture and the resulting brown suspension filtered through a pad ofsilica (300 mL), washing the pad with ether (3×100 mL). The ether phaseswere combined, concentrated, and purified to yield 5.0 g (90%) ofketone.

Synthesis of Compound 45

A 100 mL round bottom flask was charged with Compound 44 (1.4 g, 2.4mmol), 1,2,4-butanetriol (0.51 g, 4.8 mmol), pyridiniump-toluenesulfonate (0.06 g, 0.24 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (30 mL) added viacannula. The flask was equipped with a Dean-Stark tube and condenser andflushed with nitrogen. The reaction was refluxed under nitrogenovernight and progress of the reaction monitored via TLC. Afterrefluxing for three hours, reaction solution deposited in the Dean-Starktube was removed via syringe (20 mL) and the reaction vessel immediatelyreplenished with fresh toluene (20 mL). This was repeated every hour,for a total of three times, and then left to reflux mildly overnight.After cooling to room temperature, the reaction mixture was transferredto a 250 mL separatory funnel with toluene (2×5 mL), washed with 5%aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.67 g of crude product which waspurified via column chromatography on silica gel (50 g) usingdichloromethane as eluent. Yield: 1.4 g, 2.06 mmol, 86%.

Synthesis of Compound 46

A 100 mL round bottom flask was charged with Compound 45 (1.4 g, 2.06mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.72 g, 7.1 mmol, 0.99 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.74 g, 4.1 mmol) and DCM (20 mL) wasprepared. This solution was added drop wise to the above solution over a30 minute period. The reaction vessel was maintained at −15° C. Thereaction mixture was stirred at room temperature overnight and monitoredvia TLC. The reaction mixture was then diluted with DCM (25 mL), andwashed with NaHCO₃ (2×30 mL), then dried over anhydrous MgSO₄. The crudeproduct (1.7 g) was used in the next step without further purification.

Synthesis of DPan-C2K-DMA (Compound 47)

A 500 mL round bottom flask was charged with crude Compound 46 (1.7 g,2.5 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 65 mL) subsequently added via syringe.The resulting mixture was stirred for three days at room temperature.The reaction was concentrated and the crude product purified by columnchromatography using silica gel (40 g) with a gradient of 0-5% methanolin dichloromethane.

Synthesis of Compound 48

A 100 mL round bottom flask was charged with Compound 44 (1.2 g, 2.1mmol), 2-hydroxymethyl-1,3-propanediol (0.45 g, 4.2 mmol), pyridiniump-toluenesulfonate (0.05 g, 0.21 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (45 mL)subsequently added via cannula. The flask was equipped with a Dean-Starktube and condenser and flushed with nitrogen. The reaction was refluxedunder nitrogen overnight and progress of the reaction monitored via TLC.After refluxing for three hours, reaction solution deposited in theDean-Stark tube was removed via syringe (20 mL) and the reaction vesselimmediately replenished with fresh toluene (20 mL). This was repeatedevery hour, for a total of three times, and then left to reflux mildlyovernight. After cooling to room temperature, the reaction mixture wastransferred to a 250 mL separatory funnel with toluene (2×5 mL), washedwith 5% aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.44 g of crude product which was thenpurified via column chromatography on silica gel (35 g) with 0-3%methanol gradient in dichloromethane.

Synthesis of Compound 49

A 250 mL round bottom flask was charged with Compound 48 (1.2 g, 1.8mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.62 g, 6.1 mmol, 0.85 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.67 g, 3.7 mmol) and DCM (20 mL) wasprepared. This solution was added drop wise to the above solution over a30 minute period. The reaction vessel was maintained at −15° C. duringthe addition. The reaction mixture was stirred at room temperatureovernight and monitored via TLC. The reaction mixture was then dilutedwith DCM (25 mL) and washed with NaHCO₃ (2×30 mL), then dried overanhydrous MgSO₄. The crude product (1.6 g) was used in the followingstep without further purification.

Synthesis of DPan-C1K6-DMA (Compound 50)

A 250 mL round bottom flask was charged with crude Compound 49 (1.6 g,2.1 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 60 mL) subsequently added via syringe.The resulting mixture was stirred for six days at room temperature.After solvent was evaporated, the crude product was purified usingcolumn chromatography on silica gel (30 g) with 0-30% ethyl acetategradient in hexanes.

Synthesis of Compound 51

A 50 mL round bottom flask was charged with(R)-γ-hydroxymethyl-γ-butyrolactone (1.0 g, 8.6 mmol), flushed withnitrogen, and sealed with a rubber septum. Anhydrous THF (40 mL) wassubsequently added via syringe. The (R)-γ-hydroxymethyl-γ-butyrolactonesolution was then added drop wise under nitrogen to a prepared solutioncontaining LiAlH₄ (3.5 g, 92 mmol) in 160 mL anhydrous THF. During theaddition, the reaction vessel was maintained at 0° C. The resultingsuspension was stirred at room temperature overnight. The reactionmixture was cooled to 0° C. and brine (10-22 mL) added very slowly usinga Pasteur pipette. The mixture was stirred under nitrogen at roomtemperature overnight. The white solid was filtered and washed with THF(3×25 mL). The organics were combined and concentrated. After solventwas removed, the crude product seemed to contain water along with anoily residue; therefore, the crude product was azeotroped within ethanol(100 mL) resulting in a yellow oil. The crude product (0.45 g) was usedin the next step without further purification.

Synthesis of Compound 52

A 100 mL round bottom flask was charged with Compound 44 (1.0 g, 1.8mmol), Compound 51 (crude, 0.450 g, 3.6 mmol), pyridiniump-toluenesulfonate (0.05 g, 0.24 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (45 mL)subsequently added via cannula. The flask was equipped with a Dean-Starktube and condenser and flushed with nitrogen. The reaction was refluxedunder nitrogen overnight and progress of reaction monitored via TLC.After refluxing for three hours, reaction solution deposited in theDean-Stark tube was removed via syringe (20 mL) and the reaction vesselimmediately replenished with fresh toluene (20 mL). This was repeatedevery hour, for a total of five times, and then left to reflux mildlyovernight. After cooling to room temperature, the reaction mixture wastransferred to a 250 mL separatory funnel with toluene (2×5 mL), washedwith 5% aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.13 g of crude product which was thenpurified via column chromatography on silica gel (30 g) usingdichloromethane as eluent. Yield, 1.0 g.

Synthesis of Compound 53

A 250 mL round bottom flask was charged with Compound 52 (1.0 g, 1.44mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.51 g, 5 mmol, and 0.7 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.54 g, 3.0 mmol) and anhydrous DCM (20 mL)was prepared. This solution was added drop wise to the above solutionover a 30 minute period. The reaction vessel was maintained at −15° C.The reaction mixture was stirred at room temperature overnight andmonitored via TLC. The reaction mixture was then diluted with DCM (25mL) and washed with NaHCO₃ (2×30 mL), then dried over anhydrous MgSO₄.The crude product (1.2 g) was used in the next step without furtherpurification.

Synthesis of DPan-C3K-DMA (Compound 54)

A 100 mL round bottom flask was charged with crude Compound 53 (1.2 g,1.6 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 45 mL) subsequently added via syringe.The resulting mixture was stirred for four days at room temperature.After solvent was evaporated, the crude product was purified usingcolumn chromatography on silica gel (30 g) with 0-30% ethyl acetategradient in hexanes.

Example 10 Synthesis of DLen-C2K-DMA

DLen-C2K-DMA (Compound 58) having the structure shown below wassynthesized as described in Scheme 5 below.

Synthesis of dilinolenyl ketone (Compound 55)

To a 1000 mL RBF containing a solution of dilinolenyl methanol (6.0 g,11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 4.2 g (8.0 mmol, 70%) of the ketone.

Synthesis of linolenyl ketal (Compound 56)

A 100 mL RBF was charged with dilinolenyl ketone (Compound 55) (4.2 g,8.2 mmol), 1,2,4-butanetriol (3.4 g, 32 mmol), PPTS (200 mg, 0.8 mmol)and a stir bar. The flask was flushed with nitrogen and anhydroustoluene (60 mL) added. The reaction vessel was fitted with a Dean Starktube and condenser and brought to reflux and the reaction was leftovernight. After cooling to room temperature, the reaction mixturediluted with toluene (50 mL), and washed with 5% aq. Na₂CO₃ (2×50 mL),water (50 mL), dried (MgSO₄) and purified by chromatography to yield 3.0g (4.9 mmol, 59%) of the ketal.

Mesylate Formation (Compound 57)

A 250 mL RBF was charged with the ketal (Compound 56) (3.0 g, 4.9 mmol),TEA (2.2 mL, 15.6 mmol) and a stir bar. The flask was flushed withnitrogen, anh. DCM (20 mL) added, and the solution cooled to −15° C. Ina separate 50 mL flask, a solution of MsCl (9.7 mmol, 2 eqv.) inanhydrous DCM (30 mL) was prepared, then transferred to the reactionvessel by syringe over 20 minutes. The reaction was stirred for 90minutes at −15° C., at which point starting material had been consumed.The reaction mixture was diluted with a further 50 mL of DCM, washedwith NaHCO₃ (2×50 mL), dried (MgSO₄) and purified by chromatography.Final yield 3.1 g, 4.5 mmol, 92%.

Synthesis of DLen-C2K-DMA (Compound 58)

A 250 mL RBF was charged with the mesylate (Compound 57) (3.0 g, 4.35mmol), isopropanol (25 mL) and a stir bar. The flask was flushed withnitrogen, sealed, and a 2.0 M solution of dimethylamine in methanol (120mL) added via canulla. The reaction was stirred at room temperature for3 days. The solution was concentrated and purified by chromatography.Final yield 2.49 g, 3.9 mmol, 90%.

Example 11 Synthesis of γ-DLen-C2K-DMA

γ-DLen-C2K-DMA (Compound 62) having the structure shown below wassynthesized as described in Scheme 6 below.

Synthesis of di-γ-linolenyl ketone (Compound 59)

To a 1000 mL RBF containing a solution of di-γ-linolenyl methanol (6.0g, 11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 5.5 g (10.5 mmol, 92%) of ketone.

Synthesis of γ-linolenyl ketal (Compound 60)

A 100 mL RBF was charged with di-γ-linolenyl ketone (Compound 59) (2.14g, 4.1 mmol), 1,2,4-butanetriol (1.7 g, 16.0 mmol), PPTS (100 mg, 0.4mmol) and a stir bar. The flask was flushed with nitrogen and anhydroustoluene (30 mL) added. The reaction vessel was fitted with a Dean Starktube and condenser and brought to reflux and the reaction was leftovernight. After cooling to room temperature, the reaction mixture waswashed with 5% aq. Na₂CO₃ (2×50 mL), water (50 mL), dried (MgSO₄) andpurified by chromatography to yield 1.34 g (2.2 mmol, 53%) of the ketal.

Mesylate formation (Compound 61)

A 250 mL RBF was charged with the ketal (Compound 60) (1.34 g, 2.19mmol), TEA (1 mL, 7.1 mmol) and a stir bar. The flask was flushed withnitrogen, anh. DCM (10 mL) added, and the solution cooled to −15° C. Ina separate 50 mL flask, a solution of MsCl (342 μL, 4.4 mmol, 2 eqv.) inanhydrous DCM (15 mL) was prepared, then transferred to the reactionvessel by syringe over 20 minutes. The reaction was stirred for 90minutes at −15° C., at which point starting material had been consumed.The reaction mixture was diluted with a further 50 mL of DCM, washedwith NaHCO₃ (2×50 mL), dried (MgSO₄) and purified by chromatography.Final yield 1.31 g, 1.90 mmol, 87%.

Synthesis of γ-DLen-C2K-DMA (Compound 62)

A 250 mL RBF was charged with the mesylate (Compound 61) (1.31 g, 1.9mmol), isopropanol (10 mL) and a stir bar. The flask was flushed withnitrogen, sealed, and a 2.0 M solution of dimethylamine in methanol (60mL) added via canulla. The reaction was stirred at room temperature for3 days. The solution was concentrated and purified by chromatography.Final yield 1.1 g, 1.72 mmol, 91%.

Example 12 Lipid Encapsulation of siRNA

All siRNA molecules used in these studies were chemically synthesizedand annealed using standard procedures.

In some embodiments, siRNA molecules were encapsulated into serum-stablenucleic acid-lipid particles (SNALP) composed of the following lipids:(1) the lipid conjugate PEG2000-C-DMA (3-N-[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or morecationic lipids or salts thereof (e.g., cationic lipids of Formula I-XIIof the invention and/or other cationic lipids described herein); (3) thephospholipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (AvantiPolar Lipids; Alabaster, Ala.); and (4) synthetic cholesterol(Sigma-Aldrich Corp.; St. Louis, Mo.) in the molar ratio1.4:57.1:7.1:34.3, respectively. In other words, siRNA molecules wereencapsulated into SNALP of the following “1:57” formulation: 1.4%PEG2000-C-DMA; 57.1% cationic lipid; 7.1% DPPC; and 34.3% cholesterol.It should be understood that the 1:57 formulation is a targetformulation, and that the amount of lipid (both cationic andnon-cationic) present and the amount of lipid conjugate present in theformulation may vary. Typically, in the 1:57 formulation, the amount ofcationic lipid will be 57.1 mol %±5 mol %, and the amount of lipidconjugate will be 1.4 mol %±0.5 mol %, with the balance of the 1:57formulation being made up of non-cationic lipid (e.g., phospholipid,cholesterol, or a mixture of the two).

In other embodiments, siRNA were encapsulated into SNALP composed of thefollowing lipids: (1) the lipid conjugate PEG750-C-DMA(3-N-[(-Methoxypoly(ethyleneglycol)750)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or morecationic lipids or salts thereof (e.g., cationic lipids of Formula I-XIIof the invention and/or other cationic lipids described herein); (3) thephospholipid DPPC; and (4) synthetic cholesterol in the molar ratio6.76:54.06:6.75:32.43, respectively. In other words, siRNA wereencapsulated into SNALP of the following “7:54” formulation: 6.76 mol %PEG750-C-DMA; 54.06 mol % cationic lipid; 6.75 mol % DPPC; and 32.43 mol% cholesterol. Typically, in the 7:54 formulation, the amount ofcationic lipid will be 54.06 mol %±5 mol %, and the amount of lipidconjugate will be 6.76 mol %±1 mol %, with the balance of the 7:54formulation being made up of non-cationic lipid (e.g., phospholipid,cholesterol, or a mixture of the two).

For vehicle controls, empty particles with identical lipid compositionmay be formed in the absence of siRNA.

Example 13 pK_(a) Measurements of SNALP Formulations Containing NovelCationic Lipids

This example demonstrates the determination of pK_(a) values of 1:57SNALP formulations containing various novel cationic lipids describedherein with an siRNA targeting apolipoprotein B (APOB).

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLinDMA; (2) DLenDMA; (3) γ-DLenDMA (“g-DLenDMA”); (4) C2-DLinDAP; (5)DLinPip (4-OH); (6) DLinPip; (7) DLinPip (3-OH); (8) DLinDEA; (9)DDocDMA; (10) DAraDMA; (11) TLinDMA; (12) DLin-C2K Pip (30H); (13)DHep-C2K-DMA; (14) DPanDMA; (15) C2-DPanDMA (“DPan-C2-DMA”); (16)DPan-C2K-DMA; (17) DPan-C3K-DMA; (18) DPan-K6-DMA (“DPan-C1K6-DMA”); and(19) C2-TLinDMA.

The apparent pK_(a) of the cationic lipids present in these SNALPformulations was determined using a2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) assay. TNS is anegatively-charged indicator of membrane potential that iselectrostatically attracted to positively charged membranes (see, Baileyand Cullis, Biochemistry, 33 12573-80 (1994)). Subsequent adsorption tothe lipid membrane results in the immediate environment of the TNSbecoming more lipophilic, removing the water molecules that otherwisequench TNS fluorescence. As a result, TNS measures the surface potentialof the particle, wherein the more positive the surface potential, thegreater the level of fluorescence. The surface pK_(a) values of eachSNALP formulation were determined by varying the local pH in thepresence of TNS. By plotting fluorescence versus pH, the pK_(a) of thecationic lipid can be estimated in the particle as the pH wherefluorescence equals 50% of total fluorescence.

The results of the TNS assays are shown in FIGS. 1-2. FIG. 2 (top panel)shows the following 1:57 SNALP pK_(a) values: DLinDMA ˜5.8; DDocDMA,DAraDMA ˜5.65; TLinDMA ˜5.35; DLin-C2K Pip (30H), DHep-C2K-DMA ˜6.05;and C2:DPanDMA ˜6.1. FIG. 2 (bottom panel) shows the following 1:57SNALP pK_(a) values: DLinDMA ˜5.8; DPanDMA 5.5; C2-DPanDMA ˜6.1;DPan-C2K-DMA ˜5.95; DPan-K6-DMA ˜5.5; and DPan-C3K-DMA ˜6.25. The 1:57SNALP pK_(a) value for C2-TLinDMA was ˜5.85.

Example 14 Characterization of SNALP Formulations Containing NovelCationic Lipids

This example demonstrates the efficacy of 1:57 SNALP formulationscontaining various novel cationic lipids described herein with an siRNAtargeting ApoB in a mouse liver model. The APOB siRNA sequence used inthis study is provided in Table 1.

TABLE 1 % 2′OMe- % Modified siRNA APOB siRNA Sequence Modifiedin DS Region ApoB-10164 5′-AGU G UCA U CACAC U GAAUACC-3′ (SEQ ID NO: 1)7/42 = 16.7% 7/38 = 18.4% 3′-GU U CACAGUAGU G U G AC U UAU-5′(SEQ ID NO: 2) Column 1: The number after “ApoB” refers to thenucleotide position of the 5′ base of the sense strand relative to thehuman APOB mRNA sequence NM_000384. Column 2: 2′OMe nucleotides areindicated in bold and underlined. The 3′-overhangs on one or bothstrands of the siRNA molecule may alternatively comprise 1-4deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodified uridine(U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double-stranded (DS) region ofthe siRNA molecule are provided.

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLin DMA; (2) DLin-K-C2-DMA (“C2K”); (3) DLin-K-C3-DMA (“C3K”); (4)DLin-K-C4-DMA (“C4K”); (5) DLin-K6-DMA; (6) DLin-C2-DMA; (7) DLenDMA;(8) γ-DLenDMA (“g-DLenDMA”); (9) DLin-K-DMA; (10) DLinMorph; (11)Linoleyl/Oleyl DMA (“Lin/Ol”); (12) Linoleyl/Linolenyl DMA (“Lin/Len”);(13) Linoleyl/Phytanyl DMA (“Lin/Pan”); (14) Linoleyl/Stearyl DMA(“Lin/Str”); and (15) Linoleyl/C6:1 DMA (“Lin/C6:1”).

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female Balb/c mice (n=3 per group). Plasma totalcholesterol and/or liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration. For dose response studies, SNALPformulations were administered by IV injection at 0.01 mg/kg, 0.03mg/kg, or 0.1 mg/kg into female Balb/c mice (n=3 per group). Liver ApoBmRNA levels were evaluated at 48 hours after SNALP administration.

FIGS. 3-5 show a comparison of the plasma total cholesterol knockdownefficacy and/or the liver ApoB mRNA knockdown activity of each of theseSNALP formulations (Error bars=SD). FIG. 6 shows a dose responseevaluation of three different doses of SNALP formulations containingeither DLinDMA, DLin-K-C2-DMA (“C2K”), DLenDMA, or γ-DLenDMA on liverApoB mRNA knockdown activity (Error bars=SD).

These figures illustrate that a SNALP formulation containing γ-DLenDMAwas unexpectedly more potent in silencing ApoB expression compared to aSNALP formulation containing either DLinDMA or DLenDMA and produced invivo results similar to that of a SNALP formulation containingDLin-K-C2-DMA (“C2K”). These figures also illustrate that a SNALPformulation containing an asymmetric cationic lipid such asLinoleyl/Linolenyl DMA (“Lin/Len”) displayed greater ApoB silencingactivity compared to a SNALP formulation containing DLinDMA.

Example 15 Tolerability of γ-DLenDMA SNALP Formulation

The tolerability of the 1:57 γ-DLenDMA SNALP formulation was evaluatedwith a single IV bolus injection of SNALP at either 10 mg/kg, 15 mg/kg,or 20 mg/kg in Balb/c mice (n=3 per group). Liver enzyme levels weremeasured at 48 hours post-injection. FIG. 7 shows that both DLenDMA andγ-DLenDMA SNALP formulations did not significantly elevate liver enzymelevels compared to the PBS control.

Example 16 Characterization of Additional SNALP Formulations ContainingNovel Cationic Lipids

This example demonstrates the efficacy of additional 1:57 SNALPformulations containing various novel cationic lipids described hereinwith an siRNA targeting ApoB in a mouse liver model. The APOB siRNAsequence used in these studies is provided in Table 1.

1:57 SNALP formulations containing encapsulated APOB siRNA at a 6:1lipid:drug (L:D) ratio were prepared with the following cationic lipids:(1) DLinDMA; (2) Linoleyl/Linolenyl DMA (“Lin/LenDMA”); (3) DPanDMA; (4)TLinDMA; (5) Linoleyl/C6:0 DMA (“Lin/6:0”); (6) C2-DPanDMA; (7)DLin-C2K-Pip (30H); and (8) DHep-C2K-DMA.

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female Balb/c mice (n=3 per group). Livers were colletedat 48 hours after SNALP administration and liver ApoB mRNA levels wereevaluated by performing an ApoB/GAPDH QG assay. Table 2 provides acharacterization of the SNALP formulations used in this in vivo study.

TABLE 2 SNALP Size (nm) Poly Encapsulation % 1:57 DLinDMA 74.96 0.023 7977.88 0.054 1:57 Lin/LenDMA 81.12 0.023 63 98.21 0.010 1:57 DPanDMA64.84 0.063 78 65.44 0.033 1:57 TLinDMA 71.98 0.037 51 73.12 0.069 1:57Lin/6:0 101.7 0.095 78 97.64 0.119 1:57 C2-DPanDMA 95.09 0.058 90 98.260.052 1:57 DLin-C2K-Pip (3OH) 68.92 0.093 73 73.27 0.067 1:57DHep-C2K-DMA 75.29 0.034 89 79.89 0.037 1:57 DLinDMA (TFU) 82.23 0.03989 Columns 2 & 3: The bottom value in each entry corresponds to theparticle size and polydispersity observed 10 days after the formulationwas prepared.

FIG. 8 illustrates, inter alia, that: (1) DHep-C2K-DMA, which containsdouble bonds in the trans configuration thought to reduce potency, wasunexpectedly comparable to DLinDMA with respect to silencing activity;(2) C2-DPanDMA, which contains saturated fatty alkyl chains thought todecrease potency, was unexpectedly more potent compared to DLinDMA andsubstantially more potent compared to DPanDMA with respect to silencingactivity; (3) TLinDMA displayed silencing activity that was comparableto DLinDMA; and (4) Lin/LenDMA had more potent silencing activity thanDLinDMA. A similar study with 1:57 SNALP containing C2-TLinDMA showedthat C2-TLinDMA (49% knockdown) was more potent than DLinDMA (25%knockdown) with respect to ApoB silencing activity.

In another study, 1:57 SNALP formulations containing encapsulated APOBsiRNA at a 6:1 L:D ratio were prepared with the following cationiclipids: (1) DLinDMA; (2) C2-DPanDMA; (3) DPan-C2K-DMA; (4) DPan-C3K-DMA;and (5) DPan-C1K6-DMA.

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female Balb/c mice (n=3 per group). Livers were colletedat 48 hours after SNALP administration and liver ApoB mRNA levels wereevaluated by performing an ApoB/GAPDH QG assay. Table 3 provides acharacterization of the SNALP formulations used in this in vivo study.

TABLE 3 Size (nm) Poly Encapsulation % 1:57 DLinDMA 76.63 0.033 81 1:57C2-DPanDMA 85.80 0.003 88 1:57 DPan-C2K-DMA 79.06 0.020 87 1:57DPan-C3K-DMA 93.46 0.002 90 1:57 DPan-C1K6-DMA 72.78 0.031 77

FIG. 9 illustrates that C2-DPanDMA, DPan-C2K-DMA, and DPan-C3K-DMA,which contains saturated fatty alkyl chains thought to decrease potency,were unexpectedly more potent compared to DLinDMA with respect tosilencing activity. C2-DPanDMA had the greatest activity of all thephytanyl-containing cationic lipids tested.

Example 17 Characterization of Additional SNALP Formulations ContainingNovel Cationic Lipids

This example demonstrates the efficacy of additional 1:57 SNALPformulations containing various novel cationic lipids described hereinwith an siRNA targeting ApoB in a mouse liver model. The APOB siRNAsequence used in these studies is provided in Table 1.

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLin-C2K-DMA (“C2K”); (2) γ-DLen-C2K-DMA (“g-DLen-C2K-DMA”); and (3)DLen-C2K-DMA.

For dose response studies, SNALP formulations were administered by IVinjection at 0.01 mg/kg, 0.033 mg/kg, or 0.1 mg/kg into female Balb/cmice (n=3 per group). Liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration by a branched DNA assay (QuantiGene assay) toassess ApoB mRNA relative to the housekeeping gene GAPDH.

FIG. 10 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations at three different doses (Errorbars=SD), as well as the KD50 values obtained for each of theseformulations. In particular, FIG. 10 shows that a SNALP formulationcontaining g-DLen-C2K-DMA displayed similar ApoB silencing activity atall three doses and an identical KD50 value compared to a SNALPformulation containing C2K. Furthermore, FIG. 10 shows that a SNALPformulation containing DLen-C2K-DMA displayed considerable potency insilencing ApoB mRNA expression.

Example 18 Characterization of Tumor-Directed SNALP FormulationsContaining Novel Cationic Lipids

This example demonstrates the efficacy of tumor-directed SNALPformulations containing various novel cationic lipids described hereinwith an siRNA targeting polo-like kinase 1 (PLK-1) in a mouse distaltumor model. The PLK-1 siRNA sequence used in this study is provided inTable 4.

This example demonstrates the efficacy of tumor-directed SNALPformulations containing various novel cationic lipids described hereinwith an siRNA targeting polo-like kinase 1 (PLK-1) in a mouse distaltumor model. The PLK-1 siRNA sequence used in this study is provided inTable 4.

TABLE 4 % 2′OMe- % Modified siRNA PLK-1 siRNA Sequence Modifiedin DS Region PLK1424 2/6 5′-AGA U CACCC U CCU U AAA U A U U-3′(SEQ ID NO: 3) 9/42 = 21.4% 7/38 = 18.4% 3′-C U UC U A G UGGGAG GAAUUUAU-5′ (SEQ ID NO: 4) Column 1: The number after “PLK” refers to thenucleotide position of the 5′ base of the sense strand relative to thestart codon (ATG) of the human PLK-1 mRNA sequence NM_005030. Column 2:2′OMe nucleotides are indicated in bold and underlined. The 3′-overhangson one or both strands of the siRNA molecule may alternatively comprise1-4 deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodifieduridine (U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double-stranded (DS) region ofthe siRNA molecule are provided.Column 1: The number after “PLK” refers to the nucleotide position ofthe 5′ base of the sense strand relative to the start codon (ATG) of thehuman PLK-1 mRNA sequence NM_(—)005030. Column 2: 2′OMe nucleotides areindicated in bold and underlined. The 3′-overhangs on one or bothstrands of the siRNA molecule may alternatively comprise 1-4deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodified uridine(U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double-stranded (DS) region ofthe siRNA molecule are provided.

7:54 SNALP formulations containing encapsulated PLK-1 siRNA wereprepared with the following cationic lipids: (1) DLinDMA; (2) TLinDMA;(3) DLin-C1K-DMA (“DLin-K-DMA”); (4) DPanDMA; (5) DPan-C2-DMA(“C2-DPanDMA”); (6) DPan-C2K-DMA; (7) DPan-C3K-DMA; and (8)DPan-C1K6-DMA.

Each SNALP formulation was administered by intravenous (IV) injection at3 mg/kg into Scid mice containing Hep3B tumors (n=4 per group). Tumortissue was colleted at 24 hours after SNALP administration and tumorPLK-1 mRNA levels were evaluated by performing a PLK-1/GAPDH QG assay.Table 5 provides a characterization of the SNALP formulations used inthis in vivo study.

TABLE 5 Initial Mol % Composition Encaps. Finished Product PEG-lipid |Cationic | Chol | DPPC PEG-lipid Cationic Lipid (%) Z-Avg (nm) PolyEncaps. (%) 6.76 | 54.06 | 32.43 | 6.75 PEG750-C-DMA DLinDMA 75 74 0.0898 TLinDMA 70 126 0.07 100 DLin-C1K-DMA 56 84 0.05 98 DPanDMA 75 76 0.08100 DPan-C2-DMA 86 81 0.06 100 DPan-C2K-DMA 84 76 0.08 100 DPan-C3K-DMA86 81 0.06 100 DPan-C1K6-DMA 71 80 0.06 99

FIG. 11 illustrates that all of the cationic lipids tested displayedsimilar potencies in silencing PLK-1 expression. Interestingly, therewas no difference in silencing activity between DPanDMA and C2-DPanDMAusing the tumor-directed 7:54 SNALP formulation, compared to theconsiderable difference observed in the liver with 1:57 SNALP.

Example 19 Role of ApoE in the In Vitro Uptake of SNALP by Hepatocytes

This example demonstrates the requirement of ApoE for the in vitrouptake and knockdown of 1:57 SNALP formulations on ApoE−/− primaryhepatocytes.

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section V above with the following cationic lipids: (1)DLinDMA; (2) DLin-K-C2-DMA (“C2K”); (3) DLin-K-C3-DMA (“C3K”); (4)DLin-K-C4-DMA (“C4K”); (5) DLin-K6-DMA; (6) DLin-C2-DMA; (7) DLenDMA;(8) γ-DLenDMA (“g-DLenDMA”); and (9) DLin-K-DMA.

ApoE−/− primary hepatocytes were isolated and plated at 2.5×10⁴cells/well. Each SNALP formulation was pre-incubated with 50% C57(ApoE-containing) serum or ApoE−/− serum at siRNA concentrations of4000-62.5 nM for 1 hr at 37° C. Samples were then diluted to 40-0.625 nMon the hepatocytes for 24 hr. After 24 hr, cells were lysed and ApoB andGAPD mRNA levels were measured via bDNA (QG) assay.

FIGS. 12-14 show that most of the SNALP formulations tested requiredApoE for their activity. In particular, ApoE−/− serum pre-incubation orno pre-incubation resulted in similar levels of silencing activity, butthe addition of C57 serum resulted in a significant increase insilencing activity. SNALP containing either DLenDMA or γ-DLenDMA showedthe greatest levels of silencing activity when ApoE-containing serum waspresent. SNALP containing either DLin-C3K-DMA or DLin-C4K-DMA were lessdependent on ApoE compared to other formulations tested. In fact,similar activities were observed for SNALP containing DLin-C4K-DMA inboth C57 and ApoE−/− serum.

Example 20 Apolipoprotein E-Mediated Autogenous Targeting of StableNucleic Acid-Lipid Particles (SNALP)

Abstract

Using stable nucleic acid-lipid particles (SNALP) optimized for deliveryto the liver, we have previously demonstrated potent and long-lastinghepatocellular gene silencing; however, the precise mechanismscontributing to hepatocellular activity have not been fully delineated.Here we show that liver-directed SNALP take advantage of endogenousblood components that bind to receptors expressed on the surface oftarget cell populations, facilitating SNALP uptake via receptor-mediatedendocytosis. Liver-directed SNALP formulations are remodeled in theblood compartment, acquiring endogenous apolipoprotein E in the processand causing cells that express members of the low-densitylipoprotein-receptor family of cell-surface receptors to accumulateSNALP. This mechanism is clearly differentiated from active targetingtechnologies that rely on exogenous targeting ligands, and from passivetargeting approaches that rely on the inherent physicochemicalproperties of the delivery system to influence distribution and uptake.We propose the term ‘autogenous targeting’ to describe the activetargeting of drug delivery systems by the in vivo adoption of endogenoustargeting ligands.

Introduction

Small interfering RNAs (siRNA) are powerful, target-specific moleculesdesigned to suppress gene expression through the endogenous cellularprocess of RNA interference (RNAi) [1]. Since this fundamentalgene-silencing mechanism was first recognized, tremendous progress hasbeen made in developing siRNA as a novel class of therapeutic agent fora broad spectrum of disease. However, the primary barrier to realizingthe potential of siRNA-based drugs is the difficulty delivering large,unstable siRNA molecules safely, while still facilitating disease-sitetargeting and intracellular delivery of the nucleic acid [2-4]. A numberof groups have investigated alternative nucleotide chemistry as a way toimprove the pharmacologic properties of siRNA, while others haveexamined encapsulation in lipid-based carriers like liposomes or lipidnanoparticles, methods that have been used successfully forsmall-molecule drug-delivery. Typically, lipidic systems function asdrug carriers, where the solubilized drug is encapsulated in theinternal aqueous space and enclosed by liposomal lamellae. Suchformulations can be used to overcome a drug's non-ideal properties, suchas limited solubility, serum stability, circulation half-life,biodistribution or target tissue selectivity. Previous experience withconventional small-molecule drugs has shown that the drugs that benefitthe most from liposomal delivery are those that have an intracellularsite of action, are chemically labile, and are subject to enzymaticdegradation [5]. Furthermore, when siRNA-based drugs are successfullydelivered to a target cell via a liposomal delivery system, thepharmacokinetics, biodistribution, and intracellular delivery of thesiRNA payload becomes determined by the physicochemical properties ofthe carrier rather than the nucleotide sequence or chemical modificationpattern of the siRNA duplex. By using this kind of delivery system,‘active’ targeting technologies can be incorporated that make deliveryto disease sites and uptake by target cells more efficient.

Active targeting refers to processes that aim to increase theaccumulation, retention, or internalization of a drug by attachingspecific ligands to the surface of the delivery system. This differsfrom passive ‘disease site targeting’ or ‘enhanced permeability andretention’ effects (EPR), which result in the accumulation ofappropriately designed carriers in target sites such as tumor tissue[6-7]. Active targeting has been applied to liposomal small-moleculedrug formulations with some success and has generally improved thetherapeutic index of the liposomal drug when measured in preclinicalstudies. However, despite the apparent advantages associated with thesetargeting systems, certain challenges must be addressed before they canbe successfully applied to nucleic acid delivery. One challenge is withthe process of attaching the targeting ligand to the delivery system,where ligands are chemically coupled to preformed liposomes, or,alternatively, where ligand-lipid conjugates are incorporated in thefirst stages of the formulation process. These processes are limited bysuboptimal coupling efficiencies, negative impacts on nucleicencapsulation efficiency, or suboptimal presentation of targetingligands. Another challenge is, when targeting ligands are used fornucleic acid delivery, they may cause the adaptive arm of the immunesystem to react as if the targeting ligands were foreign antigens [8].This could manifest as antibody responses to the carrier, which, atbest, may attenuate the potency of subsequent doses and, at worst, mayresult in dose-limiting toxicities or hypersensitivity reactions.

To address these challenges, we developed an approach to targeteddelivery that does not rely on decorating the delivery system withexogenous targeting ligands, but instead exploits an endogenous pathwaythat facilitates receptor-mediated hepatocellular uptake. Previously, wereported that stable nucleic acid-lipid particles (SNALP) were aneffective systemic vehicle for delivering siRNA to the murine andnon-human primate liver, and we demonstrated therapeutic effects insilencing either endogenously expressed hepatocellular transcripts orexogenous viral genes [2, 9-10]. Here we describe the mechanism by whichSNALP are remodeled in the blood and acquire endogenous apolipoprotein E(APOE) in the process, which, in turn, facilitates highly efficienthepatocellular accumulation of SNALP via low-densitylipoprotein-receptor-mediated endocytosis.

Materials and Methods

Materials.

Hanks Buffered Salt Solutions (HBSS), human recombinant insulin,ethylenediaminetetracetic acid (EDTA),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Williams'Medium E (WME), (N-morpholino)propanesulfonic acid (MOPS) buffer andhepatocyte wash buffer were purchased from Invitrogen Canada Inc.(Burlington, ON, Canada). WME contained 58 μg/mL of human recombinantinsulin, 1 nM dexamethasone, and 0.1% BSA, except where specified.Rat-tail collagen, BSA, cholesterol, and dexamethasone were purchasedfrom Sigma-Aldrich (Oakville, ON, Canada). Human high-densitylipoprotein (HDL), low-density lipoprotein (LDL), and very-low-densitylipoprotein (VLDL) were purchased from Biomedical Technologies Inc(Stoughton, Mass., USA). Human intermediate-density lipoprotein (IDL)was purchased from Athens Research and Technology (Athens, Ga., USA).Human recombinant APOE2, APOE3, and APOE4 isolated from S. frugiperdawere purchased from VWR Canlab (Edmonton, AB, Canada). ³H-CHE waspurchased from Perkin-Elmer (Boston, Mass., USA).1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and biotinylatedpoly(ethyleneglycol)distearoylphosphatidylethanolamine(Biotin-PEG-DSPE), were purchased from Avanti Polar Lipids (Alabaster,Ala., USA). Streptavidin-agarose beads were purchased from ProZyme (SanLeandro, Calif., USA). 1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane(DLinDMA) and N-[(methoxy poly(ethylene glycol)₂₀₀₀)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DMA) were synthesizedas previously described [11-12]. All tissue culture incubations werecarried out at 37° C. in an atmosphere of 5% CO₂. C57BL/6 (wild type),LDLr knockout (LDLr−/−) and APOE knockout (APOE−/−) mice (The JacksonLaboratory, JAX® Mice and Services, Bar Harbor, Me., USA) were subjectedto a 1-week acclimation period prior to use. Animal studies wereperformed in accordance with the Canadian Council on Animal Care (CCAC)guidelines and followed protocols approved by the Institutional AnimalCare and Use Committee at Tekmira Pharmaceuticals Corporation (Burnaby,BC, Canada).

SNALP Formulation.

SNALP were prepared using the method previously described [10,12]. Inbrief, an ethanolic solution of lipids was mixed with an aqueoussolution of nucleic acid. The resulting SNALP were subsequentlyconcentrated, diafiltered against 20 wash volumes of phosphate-bufferedsaline (PBS) using a cross-flow ultrafiltration cartridge (GEHealthcare, Piscataway, N.J., USA) and, finally, sterile-filteredthrough Acrodisc 0.8/0.2 μm Supor filters (Pall Corp., Ann Arbor,Mich.).

SNALP-Serum and APOE Isoform Pre-Incubations.

SNALP and the relevant serum from C57BL/6, APOE−/− or LDLr−/− mice weremixed at a ratio of 7 μg siRNA per 100 μL of serum (provides for optimallevels of activity as measured by target ‘knock down’) and incubated for1 hour at 37° C. The resulting pre-incubated SNALP were diluted with PBSand serum to final concentrations of 29.3 μg/mL siRNA and 41.3% serum.

APOE isoforms were prepared in PBS at 50 μg/mL. SNALP were diluted to 50μg/mL siRNA. Equal volumes (5 μL) of SNALP and APOE solutions were thencombined and incubated for 1 hour at 37° C.

In Vitro Serum Protein Binding to Biotinylated SNALP.

SNALP were prepared containing 0.1 mol % Biotin-PEG-DSPE andpre-incubated with the serum as described above. Streptavidin-agarosebeads (50 μL) were prewashed with PBS containing 150 mM NaCl (2×500 μL)followed by PBS containing 1M NaCl (2×1 mL). SNALP (120 nmol totallipid) was added to the beads and incubated for 1 hour at roomtemperature. The beads were then washed with PBS (150 mM NaCl, 3×500μL). Proteins bound to the SNALP were visualized by PAGE analysis usinga precast 10% NuPAGE Bis/Tris gel (Invitrogen, Burlington, ON, Canada)run in 1×MOPS buffer using the XCell SureLock Gel System (Invitrogen,Burlington, ON, Canada). The agaraose beads were first suspended in asolution of 4×LDS Sample Buffer (8.75 μL, Invitrogen), 10× ReducingReagent (3.5 μL, Invitrogen) and distilled water (7.75 μL). Proteinswere denatured by heating the suspension for 10 minutes at 70° C., and20 μl, of the resulting solution was loaded onto the gel, which was runat 200 V for 50 minutes. Proteins on the gel were stained with aCoomassie Blue solution. Proteins were identified by LC/MS using anAgilent 1100 Nano-HPLC and an ABI 4000 QTrap system. Separations wereaccomplished with a reverse-phase column (Phenomenex Jupiter Proteo, 4μm, 90 A, 75 μm×150 mm) and 0-80% acetonitrile gradient.

Isolation and Identification of Proteins Binding SNALP.

The identity of human and mouse serum proteins as visualized by PAGE wasconfirmed by LC/MS. Proteins were isolated and stained with CoomassieBrilliant Blue G250. Gel slices were cut into 1-mm cubes and washedovernight in methanol/water/acetic acid (50:45:5). The samples weredehydrated with acetonitrile (ACN) and dried in a vacuum centrifuge. Theprotein in the gel pieces were reduced with 10 mM dithiothreitol (DTT)for 30 minutes, the DTT solution was removed, and the proteins werealkylated with 100 mM iodoacetamide for 30 minutes. The samples weredehydrated with ACN, rehydrated with NH₄HCO₃, and again dehydrated withACN. The gel was rehydrated with a trypsin solution (20 ng/μl, 50 mMNH₄HCO₃, 10% ACN) and incubated overnight at 37° C. Proteins wereextracted twice with ACN/water/formic acid (50/45/5), then evaporated todryness and resuspended in ACN/water/formic acid (3/95.5/1.5) foranalysis by HPLC-ESI-MS/MS.

Mass Spectrometry.

HPLC-ESI-MS/MS was performed using an Agilent 1100 Nano-HPLC and an ABI4000 QTrap system. Separations were accomplished with a reverse-phasecolumn (Phenomenex Jupiter Proteo, 4 μm, 90 A, 75 μm×150 mm) and an ACNgradient (0-80%) with a flow rate of 300 nl/min. Electrospray voltagewas 1850 V. A positive ion data-dependent MS/MS analysis was performedwith an Enhanced MS spectrum of the 400-1600 m/z range followed by anEnhanced Resolution scan to assign charge states. For each scan cycle,the top five +2 and +3 ions were subjected to collisional-induceddissociation, and mass spectra were collected with an Enhanced ProductIon scan. The fragment mass spectra were searched against IPI_Human orIPI_Mouse databases (http://www.ebi.ac.uk/IPI/IPIhelp.html), asappropriate, using Mascot (Matrix Science) and X!Tandem(http://www.thegpm.org/TANDEM/) search engines.

In Vitro LDLr-Ligand Binding Studies.

Livers from C57BL/6 and LDLr−/− mice were homogenized and cellularmembranes purified based on the methods of Kovanen et al. [13]. Proteinswere extracted from the resulting homogenate and concentrated on aDEAE-Sepharose column according to the method of Schneider et al. [14].Protein concentrations were determined using the Pierce BCA Assay.

Aliquots containing 400 μs of protein in 1× Laemmlli buffer wereseparated by 4-12% SDS-PAGE (150V for 1.5 hours). The proteins were thentransferred to Hybond-C nitrocellulose membranes using a Mini Trans BlotSystem (Bio-Rad, Mississauga, ON, Canada) (200 mA for 2 hours).Membranes were subsequently blocked with Ligand Blot Buffer A (LBB ‘A’)(20 mM Tris, 90 mM NaCl, pH 8) containing 5% BSA.

SNALP (4.45 μg siRNA) containing 0.1 mol % Biotin-PEG-DSPE werepre-incubated with serum then added to Ligand Blot Buffer B (LBB ‘B’)(20 mM Tris, 200 mM NaCl, pH 8) containing 5% BSA and used to probemembrane protein extracts isolated from C57BL/6 and LDLr−/− livers.After 2 hours at 37° C. the blots were immediately washed 3 times withLBB followed by soaking for 3 periods of 10 minutes with LBB ‘B’. Theblots were finally incubated for 1 hour at room temperature withStreptavidin-HRP diluted 1/80,000 in LBB ‘A’ containing 5% BSA. Afterimmediately washing 3 times with LBB ‘B’ followed by 3 10-minute washeswith LBB ‘B’, bound SNALP was visualized using the Amersham ECL WesternBlotting System (GE Healthcare, Piscataway, N.J., USA).

In Vitro SNALP Uptake by Primary Hepatocytes from Wild-Type and KnockoutMice.

Primary hepatocytes from C57BL/6, APOE−/−, and LDLr−/− mice wereisolated using a collagenase perfusion method. In brief, mice wereeuthanized by intraperitoneal injection of ketamine/xylazine, a catheterwas inserted into the left ventricle of the heart, and the portal veinwas severed. Livers were initially perfused with a solution of HBSS(with no calcium or magnesium salts) containing insulin (2.9 μg/mL),EDTA (0.5 mM), and HEPES (10 mM) at a rate of 10 mL/min for 3 minutes,followed by a solution of 100 U/mL of Collagenase II in HBSS (10 mL/minfor 3 minutes). Livers were then excised and submersed in WME (15 mL) ina petri dish. The hepatocytes were extracted on ice using a cellscrapper and isolated by centrifugation at 50×g for 5 minutes at 4° C.The supernatant was removed and the pellet resuspended in HepatocyteWash Medium (30 mL). The suspension was passed consecutively through 100μm and 70 μm cell strainers (BD Biosciences, Bedford, Mass., USA). Thewashing/resuspension process was repeated a further 2 times, and thefinal resuspension was with WME containing 58 μg/mL human recombinantinsulin, 1 nM dexamethasone, and 10% FBS, rather than Hepatocyte WashMedium. Hepatocytes were counted using a haemocytometer and added to12-well plates precoated with rat-tail collagen at 3×10⁵ cells/well. Thecells were incubated for 4 hours, washed with HBSS (no salts), and freshWME was added. Cells were then incubated overnight.

Preparations of ³H-CHE-labeled SNALP that had been pre-incubated inrespective sera were diluted to siRNA concentrations of 0.25 μg/mL withWME; 1 mL SNALP per well was added to primary hepatocytes. Cells wereincubated for either 4 hours or 24 hours, washed 3 times with ice-coldHESS (no salts), and lysed with PBS containing 0.1% Triton X-100.Radioactivity of the lysates was determined by liquid scintillationcounting using Picofluor 15 and a Beckman LS6500 (Beckman Instruments,CA, USA), and protein content was determined using the Pierce BCA Assay(Thermo Scientific, Waltham, Mass., USA). Uptake was determined bynormalizing radioactivity to the amount of protein per well (n=3).

In Vitro Silencing of APOB in Primary Hepatocytes (Human/Mouse).

Primary hepatocytes from C57BL/6, APOE−/−, and LDLr−/− mice were seededin 96-well Primaria plates (BD Biosciences, Bedford, Mass., USA) at2.5×10⁴ cells/well and incubated overnight. Human hepatocytes werepurchased preseeded in 96-well plates at 5×10⁴ cells/well (BDBiosciences, Bedford, Mass., USA). SNALP, pre-incubated as describedabove, were diluted to relevant concentrations in WME, added to thehepatocytes, and incubated for up to 24 hours. For time points less than24 hours, cells were then washed with HBSS, fresh media was applied, andthe plate was returned to the incubator for the remainder of the 24hours. After 24 hours, all cells were lysed using the Quantigene LysisMixture and APOB mRNA knockdown was determined using the QuantigeneAssay (Panomics, Calif., USA). Levels of APOB mRNA were compared tothose of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)housekeeping gene (n=3).

Clearance and Biodistribution.

SNALP containing tritiated cholesteryl hexadecyl ether (³H-CHE) at 1μCi/mg total lipid were prepared as described above. SNALP wereadministered to C57BL/6, APOE−/−, and LDLr−/− mice via lateral tail-veininjection at a siRNA dose of 1 mg/kg in a 200 μL injection volume. Atappropriate time points, 20 μL of blood was obtained from a tail nick,and ³H-CHE was determined in whole blood by liquid scintillationcounting using Picofluor 15 and a Beckman LS6500 (Beckman Instruments,CA, USA). At the 24-hour time point, mice were euthanized and tissueswere collected and homogenized in a FastPrep Homogenizer (ThermoScientific, Waltham, Mass., USA). Liver (100 μL) and other tissue (200μL) homogenates were assayed for radioactivity by liquid scintillationcounting with Picofluor 40. The data are presented as % injecteddose/organ (n=3).

SNALP-Mediated Silencing of APOB mRNA and Protein Expression in KnockoutMice.

C57BL/6, APOE−/−, and LDLr−/− mice were treated with SNALP as describedabove. After 48 hours, mice were euthanized and livers were collectedinto RNALater solution (Sigma-Aldrich, Oakville, ON, Canada). A portionof each liver was homogenized and APOB mRNA silencing was measured bycomparing to the GAPDH housekeeping gene using the Quantigene Assay(Panomics, Calif., USA) (n=3).

SNALP-Human Lipoprotein Pre-Incubation.

Lipoproteins were first prepared in PBS at the concentrations typicallyfound in human serum (HDL: 1.5 mg/mL, LDL: 0.65 mg/mL, IDL: 0.075 mg/mL,VLDL: 0.15 mg/mL). SNALP were diluted with PBS to 100, 50, 25, 12.5, and6.25 μg/mL siRNA. Equal volumes (5 μL) of SNALP and lipoproteinsolutions were combined prior to incubation for 1 hour at 37° C.

Results

In Vitro Serum Protein Binding.

It is understood that many drug delivery systems, including liposomes,bind a complex array of proteins upon exposure to plasma and thatliposomes with different membrane compositions have markedly differentclearance and biodistribution properties [15]. An objective of thisstudy was to determine whether any part of SNALP pharmacology is relatedto the amount and type of protein associated with SNALP upon exposure toblood.

To identify plasma proteins that bind to SNALP, we developed an in vitroStreptavidin/Biotin pull-down procedure. SNALP containing 0.1 mol %Biotin-PEG-DSPE were incubated with mouse serum. Subsequent incubationwith streptavidin-agarose beads allowed for recovery and washing ofSNALP, along with any SNALP-associated proteins, and for analysis bySDS-PAGE. The identities of adsorbed proteins were established by LC/MSand confirmed by Western blotting. When SNALP were incubated withC57BL/6 mouse serum, they associated with a number of distinct plasmaproteins, most predominantly APOE and APOA-I (FIG. 15 a). The identityof APOE was further corroborated by the results of a pull-downexperiment following incubation with APOE−/− mouse serum, in which therewas no evidence of an APOE-comigrating band adsorbing to SNALP. As withC57BL/6 serum, APOA-I from APOE−/− serum associated with SNALP, butAPOA-IV and, to a lesser degree, APOB48 also associated with the SNALPparticle following incubation in APOE−/− serum. Incubation with LDLr−/−mouse serum facilitated the adsorption of both APOE and APOA-I.

In Vitro LDLr Ligand Blotting.

The 34 kDa glycoprotein APOE, found predominantly in VLDL, HDL, andchylomicrons, is a high-affinity ligand for LDLr and other members ofthe LDLr family of cell-surface receptors [16], some of which areabundant in the liver. To assess whether SNALP-associated APOE has thepotential to facilitate LDLr-mediated binding, we conducted a ligandblotting experiment to determine if SNALP-bound APOE would be capable ofbinding to immobilized LDLr. Detergent extracts of liver membraneproteins from LDLr-expressing C57BL/6 mice and LDLr−/− mice wereseparated by SDS-PAGE and transferred onto a nitrocellulose membrane.Membranes were probed using biotinylated SNALP that had beenpre-incubated in C57BL/6 or APOE−/− serum. Membrane-bound SNALP werevisualized by adding streptavidin-HRP and ECL. Pre-incubation inAPOE-containing serum potentiated the binding of biotinylated SNALP toimmobilized LDLr (FIG. 15 b, panel 1). When SNALP were pre-incubated inserum derived from APOE−/− mice (FIG. 15 b, panel 2), or if thepre-incubation step was omitted entirely (FIG. 15 b, panel 3), bindingof SNALP to the LDLr was not observed. The role of LDLr was furthersubstantiated when we isolated liver membrane proteins from LDLr−/− miceand performed a comparable analysis. Consistent with the LDL receptorbeing the major APOE-binding protein in liver-membrane extracts, noSNALP binding to liver membrane extracts from LDLr−/− mice was observed(FIG. 15 b, panels 4 to 6).

In Vitro Uptake and Gene Silencing in C57BL/6, APOE−/−, and LDLr−/−Primary Hepatocytes.

To elucidate the mechanism of SNALP activity in hepatocytes, weconducted in vitro uptake and gene-silencing studies using primaryhepatocytes from C57BL/6, APOE−/−, and LDLr−/− mice. Hepatocytes werecultured in serum-free conditions. To facilitate interaction with serumproteins, SNALP were pre-incubated in C57BL/6, APOE−/−, or LDLr−/− serumbefore use. Hepatocytes were exposed to radio-labelled SNALP for 4 hours(FIG. 16 a) or 24 hours (FIG. 16 b). Since cells that were exposed toSNALP for 4 hours were subsequently incubated in fresh media for afurther 20 hours, both treatment groups were allowed equal time for genesilencing to manifest.

SNALP exposed to APOE-containing sera (from either C57BL/6 or LDLr−/−mice) were taken up by LDLr-expressing hepatocytes (from either C57BL/6or APOE−/− mice) to a greater degree than in LDLr−/− hepatocytes. Uptakeof SNALP following pre-incubation with APOE−/− serum was low in allhepatocyte types. While LDLr−/− hepatocytes took up little SNALP at 4hours (FIG. 16 a), prolonged exposure resulted in APOE-dependent SNALPaccumulation comparable to that achieved in LDLr+/+hepatocytes (FIG. 16b). This suggests that an APOE-dependent mechanism, possibly involvingother members of the LDL receptor family, may facilitate SNALP uptake inLDLr−/− cells. In aggregate, the results of this experiment support arole for both APOE and LDLr in hepatocellular SNALP uptake.

We conducted a parallel study to assess APOE-dependent gene silencing inprimary hepatocytes from C57BL/6, APOE−/−, and LDLr−/− mice (FIGS. 16 cand 16 d). C57BL/6 hepatocytes, which express LDLr and actively secreteAPOE protein, exhibited potent gene silencing independent of thepre-incubation conditions or the time point of analysis. Hepatocytesderived from APOE−/− mice (expressing functional LDLr) exhibitedgene-silencing levels comparable to C57BL/6 cells when exogenous APOEwas added to the tissue culture media. Finally, consistent with theSNALP uptake studies, we observed little gene silencing in LDLr−/−hepatocytes exposed to SNALP for 4 hours; however, we observedsignificant knockdown in LDLr−/− hepatocytes after 24 hours ofcontinuous exposure to SNALP in tissue culture media complemented withexogenous APOE. These results support the hypothesis that hepatocellularSNALP uptake is potentiated by an APOE-dependent LDLr-mediated uptakemechanism that can, in part, be complemented by other APOE-dependentmechanisms in the absence of LDLr expression.

Plasma Clearance and Biodistribution in APOE−/− and LDLr−/− Mice.

To further explore the role of APOE and LDLr in SNALP pharmacology, wecharacterized the blood clearance and biodistribution of ³H-CHE-labeledSNALP following intravenous administration in C57BL/6, APOE−/−, andLDLr−/− mice. FIG. 17 a illustrates the plasma clearance properties of³H-CHE-labeled SNALP after a single intravenous administration of 1mg/kg SNALP siRNA. In C57BL/6 mice, the plasma clearance half-life ofSNALP was approximately 30 minutes, while clearance of the same SNALP inAPOE−/− mice was considerably slower (half-life of approximately 4hours). This supports the hypothesis that APOE participates in, orotherwise facilitates, the rapid clearance of this SNALP formulationfrom the blood. When administered to LDLr−/− mice, SNALP exhibitedclearance properties that were intermediate between those of C57BL/6 andAPOE−/− mice. When measured 4 hours after administration, approximately30% of the injected dose remained in the circulation of LDLr−/− mice,whereas at the same time point only 5% of the injected dose remained incirculation of C57BL/6 mice. These results suggest a role for both theLDLr and APOE in the plasma clearance of SNALP.

To further characterize the influence of APOE and LDLr on SNALPpharmacology, we conducted a biodistribution study in C57BL/6, APOE−/−,and LDLr−/− mice. FIG. 17 b shows the distribution of SNALP 24 hoursafter intravenous administration. At 24 hours, 53% of the total injecteddose had accumulated in the liver of C57BL/6 mice, while considerablyless SNALP (19%) was observed in the liver of APOE−/− mice. LDLr−/− miceaccumulated an intermediate amount of SNALP in the liver (37%),consistent with the intermediate rate of clearance (FIG. 17 a).

SNALP Efficacy in C57BL/6, APOE−/−, and LDLr−/− Mice.

The implications of SNALP clearance and biodistribution were explored byconducting a correlative analysis of SNALP efficacy, as measured by APOBgene silencing following delivery of anti-APOB siRNA. In a dose-responsestudy, C57BL/6, APOE−/− and LDLr−/− mice were administered intravenousdoses of siRNA ranging from 0.25 to 18 mg/kg. APOB mRNA was measured inthe liver 48 hours after SNALP administration. In C57BL/6 mice, SNALPwere highly active, yielding 71% knockdown even at the lowestadministered dose of 0.25 mg/kg (FIG. 17 c). In APOE−/− mice, silencingof APOB mRNA was only observed at the highest dose (18 mg/kg), whileLDLr−/− mice yielded intermediate results. Significant gene silencingwas observed at all siRNA doses, further emphasizing the role of bothAPOE and LDLr in the hepatocellular uptake of SNALP.

Human Lipoproteins Can Act as an APOE Donor Potentiating SNALP Activityin Murine APOE−/− Hepatocytes.

To examine the interaction between SNALP and human lipoproteins andassess the potential role of human APOE in SNALP pharmacology, weexposed SNALP to a variety of human lipoprotein species and examined theimpact on SNALP potency in primary hepatocytes obtained from APOE−/−mice. SNALP were incubated with purified human lipoproteins at thenominal lipoprotein concentrations found in normal human serum [17-21].The resulting APOB gene-silencing activity was determined in murineAPOE−/− hepatocytes and compared to that of SNALP incubated with C57BL/6mouse serum, APOE−/− mouse serum, or human whole serum (FIG. 18 a).

Pre-incubation of SNALP with either C57BL/6 mouse serum or human wholeserum resulted in maximal potency as measured by APOB gene silencing,while pre-incubation in APOE−/− mouse serum yielded minimal activity.When SNALP were pre-incubated with human lipoproteins, the resultingactivity correlated with the relative APOE content of the lipoproteinspecies in question. APOE-poor IDL and LDL facilitated relatively modestknockdown of APOB expression in cultured hepatocytes. The APOE-richlipoproteins, VLDL and HDL, were more effective, yielding the mostpronounced gene-silencing when combined as a cocktail in thepre-incubation step.

SNALP-Mediated APOB Gene Silencing in Cultured Human Hepatocytes.

To assess the potential for autogenous targeting to human cells, weassessed the relative potency of anti-APOB SNALP in cultured humanhepatocytes. Notably, in the absence of exogenous APOE, cultured humanhepatocytes exhibited a transfection profile more similar to that ofmurine APOE−/− hepatocytes than C57BL/6 cells (FIG. 18 b). SNALP potencywas fully constituted following pre-incubation in APOE-containing serum,while it was dramatically reduced in the ‘No Serum’ samples. Thissuggests that under the conditions of this study the cultured humanhepatocytes did not secrete sufficient APOE-containing lipoproteins tocomplement serum-free media. Notably, the murine hepatocytes used inthis study were freshly isolated primary cells prepared in our ownlaboratory, while the human hepatocytes were purchased from a vendorfollowing isolation by cadaveric liver perfusion.

Purified APOE Isoforms Potentiate SNALP-Mediated APOB Gene Silencing inCultured Human Hepatocytes.

Our final study examined the effect of individual APOE isoforms onSNALP-mediated RNA interference in cultured human hepatocytes. The APOEgene is polymorphic in humans, coding for 3 main isoforms (E2, E3, andE4). Although the isoforms only differ at amino acid positions 112 and158, the physiological effects associated with each isoform can beprofound. Presence of the E4 isoform has been strongly linked toartherosclerosis [22] and Alzheimer's disease [23]. SNALP werepre-incubated individually with either individual APOE isoforms or wholeserum then used to transfect human hepatocytes at a siRNA concentrationof 0.125 μg/mL siRNA. The differences in activity between APOE isoformswere marginal, although the pattern APOE4>APOE3>APOE2 appeared to beconserved at all SNALP exposure times (FIG. 18 c). This hierarchyloosely correlates with the reported affinities of APOE isoforms for theLDLr. APOE3 and APOE4 bind to the LDLr with equal affinity and morestrongly than the APOE2 isoform [24]. Again, SNALP that were notcomplemented with purified recombinant APOE or APOE-containing serumwere unable to mediate significant APOB knockdown in human hepatocytes.

Discussion

We previously described a number of SNALP formulations optimized fordelivery to the liver that achieved potent, enduring silencing of targetgenes [2, 9, 10]. In this study, we sought to delineate the mechanism bywhich liver-directed SNALP formulations achieve high levels of potencyand answer the specific question of how SNALP are taken up by theirtarget cells. We hypothesized that the mechanism responsible forhepatocellular uptake of SNALP may involve elements of particleremodeling and/or interaction with blood components. Previous reportssuggest that, following contact with blood, neutral liposomes rapidlyassociate with a variety of serum proteins [15] includingapolipoproteins and the more abundant opsonizing proteins of thecomplement system. Other investigators have reported that theexchangeable apolipoproteins APOE and APOA-I are able to associate withliposomes [25, 26]. Further, APOE, but not APOA-I or APOA-IV, canenhance liposomal uptake in cultured HepG2 (hepatocellular carincoma)cells [27]. Yan et al. suggested that the hepatocellular uptake of emptyliposomes may involve an interaction between liposomally associated APOEand LDLr [28].

In our characterization of the uptake mechanism of liver-directed SNALP,we began by identifying the major serum proteins binding to theparticles. We developed a method for collecting and analyzingSNALP-bound proteins with SDS-polyacrylamide gel electrophoresis (FIG.15 a) and LC/MS. The major SNALP binding proteins in normal or LDLr−/−serum are serum albumin, APOA-I, and APOE. In the absence of APOE, inAPOE−/−serum, SNALP bound APOA-IV and APOB48 in addition to bindingserum albumin and APOA-1. This suggests that, of these apolipoproteinspecies, APOE has the highest affinity for SNALP, and that SNALP mayhave a limited capacity for binding apolipoproteins. In the absence ofAPOE, the available capacity for apolipoprotein binding may be utilizedby APOA-IV and APOB48, both of which are more abundant in the serum ofAPOE knockout mice than the serum of normal mice. As APOE has a highaffinity for the LDLr [16], we developed an assay to examine whether theSNALP/serum protein complexes were capable of binding to immobilizedLDLr extracted from hepatocytes (FIG. 15 b). This analysis confirmedthat SNALP bind to LDLr in an APOE-dependent manner.

To further understand the significance of these observations, weconducted SNALP uptake and efficacy experiments in primary murinehepatocytes (FIG. 16). By collecting fresh primary hepatocytes directlyfrom wild-type and knockout mice, we were able to examine the specificconsequence of removing either LDLr or APOE from the experimentalsystem. These studies showed a high degree of SNALP uptake and SNALPsiRNA-mediated gene silencing in those cells that express LDLr (C57BL/6and APOE−/− hepatocytes), provided SNALP are exposed to a source of APOE(supplied as exogenous APOE during serum pre-incubation, or endogenouslywhen secreted by APOE-expressing cells directly into the culturemedium). In instances where either APOE or LDLr were removed from thesystem, SNALP uptake and gene silencing activity was reduced.

LDLr−/− hepatocytes accumulate significant levels of SNALP in anAPOE-dependant manner (FIG. 16), albeit at a rate that was substantiallyreduced compared to LDLr-expressing cells. This suggests that otherreceptors, possibly including other members of the LDLr family, canfacilitate APOE-mediated SNALP uptake in non-LDLr-expressing cells.Wolfrum et al. postulated that once siRNA-cholesterol conjugates hadbound to APOE-containing HDL, hepatocellular uptake could be mediatedvia scavenger receptor BI (SR-BI) [29], an APOE-binding receptor broadlyexpressed in the liver [30, 31] that also seems a likely candidate forparticipation in SNALP uptake.

When our results are considered in conjunction with the serum proteinbinding assay (FIG. 15 a), it would appear that, consistent with theresults of liposomal uptake studies performed by Bisgaier et al. [27],neither APOA-I, APOA-IV, nor APOB48 (which bind to SNALP in the absenceof APOE) are major contributors to hepatocellular SNALP uptake.

The results of our in vitro analysis are also consistent with theresults of subsequent in vivo studies. SNALP uptake by the liver is mostrapid in wild-type mice expressing both the LDL receptor and APOE, asreflected by the rapid blood clearance and the high level of SNALPaccumulation. The resulting efficacy, assessed by siRNA-mediated APOBgene silencing, confirms that liver uptake involved the hepatocytes,rather than the liver macrophages alone. Accumulation and activity ofSNALP in the livers of APOE and LDLr knockout mice was comparativelydiminished, highlighting the impact of the LDLr/APOE pathway.Nonetheless, a significant amount of SNALP was shown to accumulate inthe liver of these groups, highlighting the requirement for efficientintracellular delivery of SNALP as a precursor to mediating RNAi.

In spite of the compelling data acquired from murine studies, the extentto which this uptake pathway would be available for exploitation bySNALP in other species, particularly humans, is unclear. Our final panelof experiments (FIG. 18) aimed to address this question, withencouraging results. Pre-incubation of SNALP with human serum componentsappeared to be equally successful in effecting SNALP uptake and activityin both murine and human hepatocytes. Time-course studies revealedpotent APOE-dependent gene silencing in human hepatocytes. Importantly,SNALP activity was independent of APOE isoform.

Taken together, these results demonstrate that an endogenous uptakepathway is available and can be readily exploited to directhepatocellular SNALP uptake. Liver-directed SNALP formulations areremodeled in the blood compartment, accumulating endogenous ApoE, whichserves to target SNALP for accumulation by cells that express members ofthe LDL-receptor family of cell-surface receptors. By its very nature,this targeting mechanism removes many of the hurdles presented by activetargeting strategies that must incorporate exogenous targeting ligands.Cumbersome and expensive manufacturing processes could be circumvented,as could potential immunogenic responses to exogenously suppliedtargeting ligands, which may manifest in their most benign form asattenuated potency in multiple dosing regimes or as more seriousdose-limiting toxicities, including the development of hypersensitivityreactions. This approach is clearly differentiated from active targetingtechnologies that rely on exogenous targeting ligands, and from passivetargeting approaches that rely on the inherent physicochemicalproperties of the delivery system to influence distribution and uptake.We propose the term ‘autogenous targeting’ to describe the activetargeting of drug delivery systems by the in vivo adoption of endogenoustargeting ligands.

An understanding of APOE-mediated SNALP uptake is of considerableutility in terms of further advancing this technology. Here we havedescribed some novel techniques which we have used to improve ourunderstanding of the parameters that may influence the activity ofliver-directed SNALP formulations [2, 9, 32].

The LDL receptor family contains several members, all related by commonstructural and functional domains, including the cysteine-richligand-binding domains that bind ApoB and ApoE. The most prominentmembers of the LDL receptor family are the LDL receptor, the LDLreceptor related protein (LRP1), megalin (LRP2 or gp330), the VLDLreceptor, LR11 (SorLA), apolipoprotein E receptor type 2 (apoER2, LRPs3, 4, 5, and 6), and LRP1B [33]. These receptors are expressed intissues as diverse as liver, neuron, smooth muscle, adrenal gland,kidney, lung, eye, testes, and ovary. Members of the family areimplicated in disease processes such as atherosclerotic plaqueformation, β-amyloid production in Alzheimer's disease, and growth andproliferation of various tumor types.

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It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos., are incorporated herein by reference for allpurposes.

What is claimed is:
 1. A cationic lipid of Formula VIII having thefollowing structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴, R⁵, and R⁶ are either thesame or different and are independently an optionally substitutedC₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and nis 0, 1, 2, 3, or
 4. 2. The cationic lipid of claim 1, wherein R⁴, R⁵,and R⁶ are independently selected from the group consisting of adodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety,an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety,a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienylmoiety, an icosatrienyl moiety, and a phytanyl moiety.
 3. The cationiclipid of claim 2, wherein the octadecadienyl moiety is a linoleylmoiety.
 4. The cationic lipid of claim 3, wherein R⁴, R⁵, and R⁶ arelinoleyl moieties.
 5. The cationic lipid of claim 2, wherein theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. 6.The cationic lipid of claim 1, wherein R¹ and R² are both methyl groups.7. The cationic lipid of claim 1, having a structure selected from thegroup consisting of:


8. A lipid particle comprising a cationic lipid of claim
 1. 9. The lipidparticle of claim 8, wherein the particle further comprises anon-cationic lipid.
 10. The lipid particle of claim 9, wherein thenon-cationic lipid is selected from the group consisting of aphospholipid, cholesterol, or a mixture of a phospholipid andcholesterol.
 11. The lipid particle of claim 10, wherein thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof.
 12. Thelipid particle of claim 10, wherein the cholesterol is a cholesterolderivative.
 13. The lipid particle of claim 8, wherein the particlefurther comprises a conjugated lipid that inhibits aggregation ofparticles.
 14. The lipid particle of claim 13, wherein the conjugatedlipid that inhibits aggregation of particles comprises apolyethyleneglycol (PEG)-lipid conjugate.
 15. The lipid particle ofclaim 14, wherein the PEG-lipid conjugate comprises a PEG-diacylglycerol(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or amixture thereof.
 16. The lipid particle of claim 8, wherein the particlefurther comprises a therapeutic agent.
 17. The lipid particle of claim16, wherein the therapeutic agent is a nucleic acid.
 18. The lipidparticle of claim 17, wherein the nucleic acid is an interfering RNA.19. The lipid particle of claim 18, wherein the interfering RNA isselected from the group consisting of a small interfering RNA (siRNA),an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), aDicer-substrate dsRNA, a small hairpin RNA (shRNA), and mixturesthereof.
 20. The lipid particle of claim 18, wherein the interfering RNAis an siRNA.
 21. The lipid particle of claim 16, wherein the therapeuticagent is not substantially degraded after incubation of the particle inserum at 37° C. for 30 minutes.
 22. The lipid particle of claim 16,wherein the therapeutic agent is fully encapsulated in the particle. 23.The lipid particle of claim 17, wherein the particle has a lipid:nucleicacid mass ratio of from about 5:1 to about 15:1.
 24. The lipid particleof claim 8, wherein the particle has a median diameter of from about 30nm to about 150 nm.
 25. A pharmaceutical composition comprising a lipidparticle of claim 8 and a pharmaceutically acceptable carrier.
 26. Amethod for introducing a therapeutic agent into a cell, the methodcomprising: contacting the cell with a lipid particle of claim
 16. 27.The method of claim 26, wherein the cell is in a mammal.
 28. A methodfor the in vivo delivery of a therapeutic agent, the method comprising:administering to a mammal a lipid particle of claim
 16. 29. The methodof claim 28, wherein the administration is selected from the groupconsisting of oral, intranasal, intravenous, intraperitoneal,intramuscular, intraarticular, intralesional, intratracheal,subcutaneous, and intradermal.
 30. The method of claim 28, wherein themammal is a human.
 31. A method for treating a disease or disorder in amammal in need thereof, the method comprising: administering to themammal a therapeutically effective amount of a lipid particle of claim16.
 32. The method of claim 31, wherein the disease or disorder isselected from the group consisting of a viral infection, a liver diseaseor disorder, and cancer.
 33. The method of claim 31, wherein the mammalis a human.